Recent advances in elucidation of biological corrinoid functions

Transcription

1 FEMS Microbiology Reviews 12 (1993) Federation of European Microbiological Societies /93/$15.00 Published by Elsevier 349 FEMSRE Recent advances in elucidation of biological corrinoid functions Erhard Stupperich Department of Applied Microbiology, University of Ulm, Ulm, FRG (Received 9 June 1993; accepted 16 July 1993) Abstract: Eleven adenosylcorrinoid-dependent rearrangements and elimination reactions have been described during the last four decades of vitamin B12 research. In contrast, only the cobamide-dependent methionine synthase was well established as a corrinoid-dependent methyl transfer reaction. Yet, investigations during the last few years revealed nine additional corrinoid-dependent methyltransferases. Many of these reactions are catalyzed by bacteria which possess a distinct C l metabolism. Notably acetogenic and methanogenic bacteria carry out such methyl transfers in their anabolism and catabolism. Tetrahydrofolate or a similar pterine derivative is a key intermediate in these reactions. It functions as methyl acceptor and the methylated tetrahydrofolate serves as a methyl donor. Key words: Methanogenic bacteria; Acetogenic bacteria; Methanol; Demethylation; Ether cleavage; Dechlorination Introduction The biological function of cobalt is mainly associated with corrinoids. Until now there are only three verified exceptions of this rule, lysine 2,3-mutase [1], nitrile hydratase [2], and methionine aminopeptidase [3], which contain constitutively cobalt in a non-corrinoid from. The corrinoids, e.g. vitamin B12, are complex tetrapyrroles [4-6]. They participate as cofactors of corrinoiddependent reactions in prokaryotes and eukaryotes [7,8]. The reactions are grouped into methyl transfers (Fig. 1), which are one subject of this Correspondence to: E. Stupperich, Angewandte Mikrobiologie, Albert-Einstein-Allee 11, D Ulm, FRG. CH30.R? b CH3Hg+ ] ~ ~ CH3-SH and CH3"S'CH 3 CH3OH ~ CH T~ --~'~~ CH3.SH ~ ~ " CH3COOH ch i... 3 ~ CH3-CoM CH 4 [] Methionine ~ ~ l, CH3+ "ATP" 5-Methylguanine Fig. 1. Corrinoid-dependent methyl transfer reactions in prokaryotes and eukaryotes. The reactions are combined in three parts representing a physiological group of organisms. (A) Acetogenic bacteria; (B) methanogenic bacteria; (C) eukaryotes and some prokaryotes. CH3-THF, methyltetrahydrofolate or an analogue. 'ATP', energy-consuming or energy-releasing reaction.

2 350 article, and into C-C rearrangements or eliminations. In general, these catalyses make use of the exceptional chemical corrinoid properties. Although some of the corrinoid-dependent reactions were discovered several decades ago, their reaction mechanisms and the interactions of the corrinoid cofactor with the apoproteins are still puzzling. Several reasons might have affected this gap in our knowledge. (i) Vitamin B~2 is one of the most complex cofactors in nature. Usually, the cofactor is found in low concentrations in biological samples and therefore laborious methods are required for its analyses. (ii) Biologically important corrinoid derivatives are light-sensitive and others like the Co(I) corrinoids are unstable under oxidized conditions. (iii) There is a lack of suitable biological corrinoid analogues, because similar chemical structures were found in most corrinoids. Analogues, however, could be useful in elucidating reaction mechanisms. (iv) Most corrinoid-dependent reactions, in particular corrinoid-containing methyltransferases are catalyzed by strictly anaerobic bacteria. The mass culture of these organisms and biochemical investigations of their central reactions were hampered by their oxygen sensitivity. Some of the above-mentioned experimental limitations have been overcome during the last few years. Notably, the mass culture of anaerobic bacteria and the application of advanced spectroscopic methods to anaerobic enzymes were improved. In addition, recombinant techniques for overexpression of corrinoid-dependent enzymes are already applied successfully to corrinoid-containing proteins, e.g. the corrinoid/iron-sulfur protein from Clostridium thermoaceticum [9] or ethanolamine ammonia lyase from Salmonella typhimurium [10]. This progress has made available large-scale corrinoid protein preparations for biochemical investigations using EXAFS, EPR and NMR spectroscopy, as well as X-ray defraction analysis of protein crystals. In this respect, acetogenic and methanogenic bacteria have been investigated in detail. These strictly anaerobic bacteria catalyze the final steps in the mineralization of degradable polymers in nature. They possess a distinct C~ metabolism entailing considerable concentrations of corri- holds. Hence, studies on acetogenesis and methanogenesis resulted in exciting publications regarding their corrinoid-containing enzymes, and therefore corrinoid-dependent methyl transfers from acetogens and methanogens are the main subject of this paper. Another topic derives from the fact that only bacteria produce corrinoids. Eukaryotes have to take up corrinoids from their diet, because they catalyze corrinoid-dependent reactions although they are unable to synthesize this cofactor. Therefore, eukaryotes depend on the vitamin B j2 biosynthetic properties of some bacteria [11]. They have developed a highly efficient corrinoid uptake and transport system in order to supply the potential cofactor to their effector cells which contain the cytoplasmic methionine synthase or the mitochondrial methylmalonyl-coa mutase apoenzymes. The corrinoid recognition mechanisms of these corrinoid-binding and corrinoidtransporting proteins are not yet understood. Information about these binding mechanisms might offer interesting insights into conformation of their binding sites and into the structural features of the active site of corrinoid-containing proteins. This article is arranged into four major parts: (i) the chemical properties of corrinoids; (ii) the reactions using methyl-tetrahydrofolate or analogous compounds as methyl donors in methionine biosynthesis, methane or acetyl-coenzyme A formation, (iii) the methyl-corrinoid-dependent reactions forming CH3-tetrahydrofolate; and (iv) miscellaneous corrinoid-dependent reactions, including the human cobalamin uptake system. Chemical properties of corrinoids Corrinoids are tetrapyrroles with optimized complexations for metals [5]. In contrast to porphyrins, the corrin ring shows a contraction and conformational flexibility [12] that might improve their physiological function [13]. The chemical properties of corrinoids are reviewed most recently by Pratt [14]. The cobalt nucleus of the corrin ring occurs in three redox states, Co(III), Co(II) and Co(I). The metal ion of the diamagnetic Co(III) form is

3 351 six-coordinated by four equatorial nitrogens of the tetrapyrroles and by two axial ligands. The lower Coa-ligand is frequently a N-heterocyclic base of a nucleotide function, whereas the upper Co/3-1igand from biological sources is either a methyl, a water, or a 5'-deoxyadenosyl group. These macromolecules are so-called 'complete' corrinoids or cobamides to distinguish them from 'incomplete' corrinoids or cobinamides which lack a Coa-nucleotide loop. For the nomenclature of corrinoids see the recommendation of the Commission on Biochemical Nomenclature (JUPAC- JUB) [15]. Methyl-cob(II1)alamin is an important biological corrinoid. This molecule has been extensively investigated by various techniques. X-ray defraction analyses of methyl-cob(lli)alamin indicates an in-plane binding of the cobalt, and axial bond lengths of 1.99 A and 2.19 A for the Co-CH 3 and the Co-N coordinations, respectively [4]. Studies on the coordination chemistry of the lower Coo ligand revealed that the heterocyclic base 5,6-dimethylbenzimidazole is detached on protonation to the 'base-off' position. The pk a for the protohated nucleotide loop is [14] or 2.89 as determined by heteronuclear NMR spectroscopy [16]. The tendency to form a base-off corrinoid is affected by the upper Coil ligand. A pk a of -2.4 was determined for the protonated Coo ligand in case a water molecule is the upper ligand. The pk a shifts to 2.7 when water is replaced by an electron-donating Co/3-methyl ligand. The abstraction of the Coil-methyl group from the cobalt requires a bond dissociation energy of 150 kj mol-1 when the corrinoid is in the base-on form. This energy barrier is lowered to 50 kj moll for a one-electron reduction of the Comethyl bond. However, this electrochemical reaction is presumably not relevant to biological systems due to the lack of suitable physiological reductants that would require redox potentials even more negative than mv (vs. SCE) [171. Methyl-cobalamin and CH3-cobinamides were studied using 13C-NMR spectroscopy [18]. It is of interest that the reductive alkylation of diaquocobinamide gave rise only to the Coil-Ell 3 cobi- namides. The Coo-methyl isomer, however, could not be detected, indicating that the upper Coil face is the preferred alkylation side of incomplete corrinoids [18]. The NMR data of this Coil-methyl cobinamide are nearly identical to those obtained for the base-off species of the analogous CH 3- cobalamin [18]. The influence of the CH3, H20 or CN Co/3 ligands, and the base-on/base-off configuration of the Coo ligand on the electronic corrinoid structure were investigated also by using 15N- NMR spectroscopy [19]. The NMR technique was applied to the completely ~SN-labeled vitamin Bl2 analogue, factor III, i.d. 5-hydroxybenzimidazolyl cobamide. All cobalt-coordinated N atoms were deshielded when the Coil ligand was changed from CN to methyl. Adding an electron-donating ligand such as a methyl group to the cobalt centre weakens the nitrogen-complexing cobalt bonds, including the Co-N bond of the nucleotide loop [19]. These data are consistent with the thermodynamic 'trans effect' which was demonstrated also by the rapid equilibration of CH3-cobalamin with nucleotide-free cobinamides in anaerobic solutions at room temperature [20]. The alkyl group of CH3-corrinoids could be transferred to thiols, in particular from a base-off CH3-cobalamin [21]. This reaction could proceed mechanistically via a heterolytic or homolytic cleavage of the Co-methyl bond. Experimental data suggest that a heterolytic cleavage of the Co-CH 3 is likely with Co(I) and CH~ as intermediates [22]. In case a nucleotide-free corrinoid is methylated, the methyl group attaches predominantly to the Co/3 face of the corrin ring [22,23]. A similar alkyl corrinoid to CH3-cobalamin is 5'-deoxyadenosyl-cobalamin (Ado-Bi2), which is referred to as a 'reversible free radical carrier' [7]. The photolysis product of Ado-cob(III)alamin is the pentacoordinated and paramagnetic low spin cob(ii)alamin [24]. This cob(ii)alamin is an excellent radical trap. It is an intermediate in Ado-cobalamin-dependent C-C rearrangements and elimination reactions [25-30], which are mechanistically still puzzling [31]. The reduction of Co(Ill) to Co(II), however, hardly influences the cobalt-corrin portion of the molecule as deduced from crystallographic data, except that the

4 352 cobalt is located 0.12 A below the corrin plane [32]. Considerable conformational changes were observed in the ribosephosphate segment, owin~g to a shorter Co-N bond of the nucleotide: 2.13 A instead of 2.24 A in the Ado-Bl2 and 2.19,~ in CH3-cobalamin [32]. These data were confirmed with extended X-ray absorption fine structure (EXAFS) spectroscopy of cob(ii)alamin solutions [33]. One-electron reduction of cob(ii)alamin leads to the formation of the square-planar fourcoordinated cob(i)alamin [34]. The reduction is facilitated in a base-off CH3-cob(II)alamin rather than in a base-on CH3-cob(II)alamin, because the nucleotide coordination strengthens the Co-CH 3 bond against a CH~ abstraction by a nucleophile. Cob(I)alamin itself is one of the strongest nucleophiles in nature [35]. It has been also prepared by the reduction of hydroxycob(iii)alamin with formate [36]. Based on kinetic isotope effects, the proposed mechanism for this reduction features an internal hydride shift within a Co(III)-formate complex to yield a protonated Co(I) intermediate. O / \ Co(III) C-~-O / > Co(I)H +CO 2 H Co(I)H ~ Co(1) +H + Additional evidence for a so-called hydridocobalamin was derived from UV-visible spectroscopic analyses of cob(i)alamin solutions [37]. Protonated cob(i)amides should be stable in acidic solutions of ph < 1 [38]. Cob(I)alamin is a one-electron reducing agent with a redox potential of -740 mv (vs. SCE) for the Co(II)/Co(I) couple [38]. It is presumably involved as a catalyst of C-C bond formations in preparative organic chemistry [39,40] and in nonenzymic dehalogenations of chlorinated aryl and alkyl compounds [41-43]. One physiological function of cob(1)alamin is that of a methyl acceptor [23]. Methionine synthase The prokaryotic methionine biosynthesis and the eukaryotic salvage pathway for methionine formation employ the corrinoid-dependent homocysteine: NS-methyl-tetrahydrofolate methyltransferase (EC ). CH 3-THF + homocysteine ~ THF + methionine The Escherichia coli and the mammalian enzyme utilizes mono- and polyglutamate forms of CH 3- tetrahydrofolate as methyl donor. Both methyl transferases need a reductive activation by AdoMet (SAM) for catalyses [47]. Glutathionylcobalamin stimulated the formation of CH 3- cobalamin ten-fold and that of Ado-cobalamin four-fold, suggesting that this corrinoid or a closely related thiol-cobalamin might be a precursor in coenzyme formation [47]. The redox potential of the cob(ii)alamin/ cob(i)alamin cofactor was -526 mv (vs. SHE) in their base-on form, which favors cob(ii)alamin formation under assay conditions at -350 mv [47]. Yet, the addition of CH3-tetrahydrofolate facilitates the reduction of cob(ii)alamin since it increased the redox potential by 80 mv. On the addition of AdoMet, this reduction became irreversible by lowering the cob(ii)alamin concentration by a factor of [47]. Thus, it was proposed that cob(ii)alamin may react first with AdoMet in a homolytic methyl transfer reaction and that the reducing system is required for the subsequent reduction of the Ado-homocysteine radical cation [47]. This would be an example of a coupled reaction where a difficult reduction is linked to a methylation reaction with a large free energy decrease. The CH3-Bz2 enzyme is the starting complex after reductive methylation to which CH3-tetrahydrofolate and homocysteine bind in a sequential manner. This enzyme complex is then regenerated by the release of methionine and tetrahydrofolate. An enzyme-bound cob(i)alamin is presumably a kinetically competent intermediate during this reaction [47]. A turnover number of 1500 min-l was determined for the E. coil methionine synthase [49]. Transalkylation takes place from methyl-tetrahydrofolate to the nucleophilic Co(I) corrinoid cofactor. CH 3-tetrahydrofolate is a tertiary amine which needs an activation before the nucleophilic attack of a cob(i)alamin could occur [50]. It seems

5 353 reasonable to assume a protonated NS-methyl-te - trahydrofolate, although this activation demands a discrimination between the methyl and the proton at the folate. The problem is overcome by the strong nucleophilicity of cob(i)alamin, which is an extremely weak base with a pk a of 1. In addition to the corrineid-dependent methionine biosynthesis, a corrinoid-independent enzymic step (EC ) is known with virtually the same reactants [51,52]. Its corrinoid-independent methionine synthase contains a cysteine residue which participates as an intermediate methyl acceptor in catalysis [49]. In contrast to the corrinoid-dependent enzyme, the corrinoidindependent activity strictly required tetrahydrofolate in the polyglutamate form, Mg 2+ or Mn 2+ and phosphate ions [49]. A turnover number of 12.3 min -1 was determined for the corrinoid-independent methionine synthase [49]. Irreversible inhibition of the corrinoid-dependent methionine synthase is achieved by N20 [53]. N20 presumably oxidizes the super-reduced cob(i)alamin cofactor by the formation of HO radicals according to the following reactions: Cob(I) + N20 + H+~ Cob(II) + N 2 + OH" N20 + e-+ H20 ~ Nz + OH-+ OH" The hydroxyl radicals oxidize transitional metal complexes and prevent the formation of the active CH3-cobalamin [54]. Chloroform or carbon tetrachloride also inhibited the corrinoid-dependent methionine synthase. This was deduced from growth experiments with methionine auxotrophic and prototrophic E. coli strains [55]. The chlorinated hydrocarbons inhibited the auxotrophic cobalamin-requiring strain (ATCC 14169) in the absence, but not in the presence of k-methionine, whereas growth of the prototrophic E. coli (ATCC 14948) with the corrinoid-independent methionine synthase was not affected [55]. The finding suggested that the halogenated C 1 compounds are selective inhibitors of the bacterial methionine synthase. Other halogenated compounds which covalently modify cob(i)alamin in vitro were inactive [55], except for the alkylating propyl iodide which specifically inactivates this methyl transferase [56]. Methyl transferase activity could be restored by light, due to the photolysis of propyl-cobalamin. Protein features The corrinoid-dependent methionine synthase is a homomeric protein consisting of about 1200 amino acid residues with a relative molecular mass of 133 K, 1 mol copper and 1 mol corrinoid per mol protein [52]. The enzyme occurs in bacteria and in the cytoplasm of mammalian cells. The human apoenzyme is converted into the holoenzyme by the incorporation of a cobalt(ii) corrinoid [57]. Remarkably, full activation of the human methionine synthase was achieved with different corrinoid forms, e.g. with HO, CN or CH 3 Co/3 ligands, deaminated or methylated acetamide and propylamide side chains of the corrin ring, or modified Cod ligands. Cobinamide for instance, the nucleotide-free corrinoid, was even more efficient in the activation of methionine synthase than the complete cofactor cobalamin [57]. This observation is consistent with the finding that the base-off CH3-cobalamin was more easily cleaved heterolytically to Co(I) and CH 3+ than the base-on form [47]. Limited tryptic digestion of the E. coli holoenzyme resulted in loss of enzyme activity, but retention of bound cobalamin to a 28-kDa peptide. This fragment extend from residue 643 to 900 of the l124-residue deduced amino acid sequence [58]. A similar fragment was obtained during crystallization of the holoenzyme at room temperature due to a proteolytic digestion during this procedure. The 28-kDa fragment was crystallized and a native X-ray defraction data set was collected to 3 A resolution [59]. Molecular biological analyses of methionine synthase The methionine biosynthesis is encoded by the met genes, which are scattered throughout the E. coli chromosome [60]. The meth and the mete genes encode the corrinoid-dependent (EC ) and the corrinoid-independent (EC ) methionine synthase, respectively. The deduced amino acid sequence of both proteins revealed an absence of similarity [49].

6 354 The methionine biosynthetic genes are controlled by three distinct types of regulation: (i) they are repressed, when the bacterium grows in the presence of methionine; (ii) the meth and the mete genes are activated by a metr gene product, which is modulated by homocysteine; and (iii) the mete gene is repressed by vitamin B12 [60-62]. The molecular mass of the meth gene product was calculated to Da [63]. A comparison of the meth nucleotide sequence from E. coli with that of the partially sequenced gene from Salmonella typhimurium revealed 92% homology of the first 414 amino acids [63]. A significant homology of the deduced amino acid sequence was not found with that of other corrinoid-containing proteins. Physiological role of methionine Methionine is a precursor of the essential S- adenosyl methionine (AdoMet or SAM), which is formed after an ATP-dependent activation in the SAM synthetase reaction. SAM is a central CH3-donor of methyltransferases in prokaryotes [64,65] and in eukaryotes since the methyl group of the charged sulfonium ion is a proper leaving group. This fact is used for instance in the biosyntheses of corrinoids, which proceed via the uroporphyrinogen III methyltransferase from uroporphyrinogen III to the precorrin series [66]. This methyltransferase was purified from Propionibacterium denitrificans in the course of a molecular biological approach to corrinoid biosynthesis [67,68]. A different SAM-dependent reaction mechanism is the activation of lysine 2,3-aminomutase. The corrinoid-independent enzyme accepts the 5'-deoxyadenosyl moiety at a not yet identified cofactor [69]. Corrinoid-dependent reactions involved in methane formation Two corrinoid-dependent reactions lead to the formation of methyl-coenzyme M (CH3-SCoM), which is the substrate of the methane-forming methyl-com methyl reductase. The specific methanogenic cofactor 2-mercaptoethanesulfonate (SCoM) [70] is methylated with the methyl donors methanol [71] or with NS-methyltetrahy - dromethanopterin [72]. The last reaction resembles the methionine biosynthesis in that a methylated pterine donates the methyl group and a thiol is the methyl acceptor. However, the reaction mechanism in methanogens appears to be even more complicated than the methionine synthase reaction. All corrinoid-dependent enzymes are located within the cytoplasm of prokaryotic and eukaryotic cells, with the exception of an unusual corrinoid-containing membrane protein from methanogenic bacteria [73-75]. This protein is presumably involved in the exergonic methyltransfer (AG o' = - 30 kj mol- 1) from NLmethyltetrahydromethanopterin to methyl-coenzyme M [76,77]. The following experimental data support this hypothesis. A corrinoid-containing membrane protein from Methanobacterium thermoautotrophicum strain Marburg was characterized by its N-terminal amino acid sequence and by monospecific polyclonal antibodies [78]. The antibodies were tested for cross-reactivities with various corrinoid-containing proteins, but showed cross-reactions only with a 33-kDa membrane protein and a 33-kDa methyltransferase from the cytoplasmic fraction of M. thermoautotrophicum strain AH [78]. It was indicative that the membrane protein and the cytoplasmic protein from strain AH are similar to the corrinoid-containing membrane protein from strain Marburg, since not only the soluble methyltransferase but also the membrane fraction of M. thermoautotrophicum strain zlh showed methyltransferase activity [79]. Indeed, a membrane protein complex was isolated from M. thermoautotrophicum strain Marburg [80], which revealed NS-methyltetrahydro - methanopterin :coenzyme M methyltransferase activity [81]. This protein complex was resolved into seven subunits including a corrinoid-containing peptide which exhibited an identical N-terminal amino acid sequence as published previously [78]. Molecular biological analyses of the corrinoidcontaining membrane protein revealed a 3.5-kb EcoRI fragment from the M. thermoautotrophicum chromosome that consists of four complete

7 355 and one incomplete open reading frames, genes mtra to mtre [82]. MtrA encodes a hydrophilic gene product of 100% identity with the first 26 N-terminal amino acids of the corrinoid-containing membrane protein [78], a tentative membrane-spanning region near the N-terminus and a hydrophilic core of the protein, which projects antigenic determinants into the cytoplasm [82]. The extreme hydrophobic gene products of mtrc and mtrd were identical to the N-termini of two peptides enriched with the functional methyl transferase complex [81]. One of these hydrophobic gene products might function as a permease, for instance as a Na+/H + antiporter [83], since the membrane-bound methyl transferase activity of Methanosarcina G61 was found to be a sodium-translocating protein [84,85]. The mechanism of coupling the methyl transfer to a sodium translocation is presently under investigation. Figure 2 depicts a tentative scheme which takes into consideration the deduced amino acid sequence of the corrinoid-containing membrane protein [82]. Remarkable charge clusters at the methyl transferase C-terminus could be involved in mediating a cation translocation, whilst the extreme hydrophobic tail of the corrinoid protein contacts a hydrophobic permease [82]. A translocated proton could activate the proteinbound CH3-tetrahydromethanopterin analogous to the methionine synthase substrate. A similar Na+/H + antiporter-methyl transferase reaction may occur in acetogenic bacteria [86]. The molecular biological analyses of the mtr gene cluster also indicate that the membranebound methyl transferase should exists of at [east five subunits with a relative molecular mass of > 110 kda. The enzyme complex catalyzes the formation of CH3-SCoM, the substrate of the methane-forming methyl-com methyl reductase. The methyl-com reductase (mcr) genes are located on the Notl, Pmel and Nhel fragments A of the Methanobacterium chromosome [87]. Interestingly, the mtr gene cluster also hybridized to these fragments A (R. Stettler and T. Leisinger, personal communication), where all the genes of methanogenesis have been located so far. Another corrinoid-dependent reaction in methanogenic bacteria is involved in the methanol Na + AI, Permease ~ ~ NH2,~) Periplasm Membrane o( ~ C~toplasm co CH3, HIMPT ~ HS-CoM Fig. 2. Tentative scheme for coupling a Na+/H + translocation to the NS-methyltetrahydromethanopterin:coenzyme M methyltransferase activity. A negative charge cluster at the C-terminus of the protein might be involved in cation translocation. An activation of methyltetrahydromethanopterine by a proton could occur in a reaction analogous to the methyl donor activation by the methionine synthase. metabolism of Methanosarcina barkeri [88-90] according to the following equation [91]: 4CH3OH ~ 3CH 4 + CO 2 + H20 AG o' = kj (mol CH4) -I The methanol methyl is transferred to S-CoM, a specific methanogenic cofactor, by the action of the methanol : 5-hydroxybenzimidazolyl cobamide methyltransferase and an additional corrinoid-independent transferase. The corrinoid-containing methyl transferase has an estimated molecular mass of 122 kda and consists of an a2/3 subunit structure. 3-4 mol corrinoid per mol protein have been determined which could be removed by treatment of the enzyme with 2-mercaptoethanol or sodium dodecyl sulfate [89]. Divalent cations, H e and hydrogenase, catalytic amounts of ATP and an oxygen-labile activation protein were required for methyl transferase activity [92,93].

8 356 Corrinoid-dependent reactions in acetate metabolisms and dehalogenations Acetyl-coenzyme A biosynthesis in acetogenic bacteria [94] as well as the acetate catabolism of methanogenic bacteria [95,96] involves similar corrinoid-dependent methyltransferases (Fig. 3). An unusual 88-kDa corrinoid/iron-sulfur protein was purified as an a/3-dimer from the carbon monoxide dehydrogenase complex of Clostridium thermoaceticum [97]. The 33-kDa subunit contains the corrinoid cofactor 5-methoxybenzimidazolyl cobamide, and the 55-kDa subunit carries an [4Fe-4S] 1+/2+ cluster. This enzyme mediates the methyl transfer from CH3-tetrahydrofolate to a nucleophilic center located on the carbon monoxide dehydrogenase [98]. EPR studies on the corrinoid/iron-sulfur protein indicated that the corrinoid cofactor was in the base-off conformation during catalyses, independent of its redox state [97]. This finding CH3-TH F Corrinoid j l" membrane-bound, Na+-translocation t CH3-S-CoM CH3 "H4SPT E0= "504 mv t [ Corrinoid/Fe-S ] E0 :-486 mv base-off T CO-DH *--- CoA + CO *-~ CO-DH base-off I ~ Ac-CoA -- S-CoM acetogens methanogens Fig. 3. Corrinoid-dependent reactions in acetogenic and methanogenic bacteria. Ac-CoA, acetyl-coenzyme A; CH 3- THF, methyltetrahydrofolate; CH3-H4SPT, methyltetrahydrosarcinapterine; S-CoM, 2-mercaptoethanesulfonic acid; CO-DH, carbon monoxide dehydrogenase. was confirmed by X-ray absorption spectroscopy of the inactive enzyme, which showed a four-coordinated Co(II) state [99]. The four-coordinated Co(II) state is an unusual coordination because the base-off corrinoid in solution is only stable below ph 3. It was pointed out that the four-coordinated cob(ii)amide cofactor poises the Co(II) state for facile reduction to a four-coordinated Co(I). EPR and UV-visible spectroscopy showed that the midpoint reduction potentials of the corrinoid/iron-sulfur protein was mv (vs. SHE) for the Co(III)/Co(II) and -504 mv for the Co(II)/Co(I) couples [100]. The corrinoid cofactor 5-methoxybenzimidazolyl cobamide in solution revealed a more negative redox potential of +207 mv for the Co(III)/Co(II) couple, indicating that the corrinoid binding to the protein facilitates the cofactor reduction. The [4Fe- 4S] 1+/2+ carrying subunit mediates the carbon monoxide-dependent reduction in vivo [98]. A gene cluster encodes the corrinoid/iron-sulfur heterodimer that was sequenced and expressed as a recombinant protein with enzymic activity [9]. Methanothrix and Methanosarcina species utilize acetate in methane formation [95,96,101] at divers acetate affinities [102]. The reaction proceeds according to the following equation [95]: CH3COO + H+-* CH 4 + CO 2 AG ~v= -36 kj (mol acetate) 1 The enzymology of this reaction resembles in part the acetyl-coa formation in acetogenic bacteria (Fig. 3). Acetate metabolism of methanogens is dependent on ferredoxin [103] and CoA [104], since acetyl-coa is the physiological substrate [105]. A propyl iodide inhibition of this reaction was an early indication for a corrinoid-dependent pathway [106]. Indeed, two corrinoid-containing enzymes became labelled when [14C]acetatc was used [107] in the intermediary formation of methyl-tetrahydrosarcinapterin [108]. Ac-CoA + H4SPT + H20 -~ CH3-SPT + CO 2 + 2[H] + CoA The key reaction of this sequence is catalyzed by the carbon monoxide dehydrogenase that cleaves

9 357 acetyl-coenzyme A into the methyl, carbonyl and the CoA components. The methanogenic enzyme resembles the acetogenic CO-DH in that it consists of a Ni/Fe-S and a corrinoid-fe-s component [109]. The methanogenic corrinoid cofactor is maintained also in the base-off state during catalysis and it showed an equilibrium reduction potential (E ') of -486 mv (vs. SHE) for the couple Co(II)/Co(I) and of -502 mv for the [4Fe-4S] 2+/1+ cluster at ph 7.8 [109]. The structural diversities of the methyl acceptors tetrahydromethosarcinapterin, tetrahydromethanopterins and tetrahydrofolates have been discussed elsewhere [110]. It was found that corrinoid solutions are useful catalysts in organic synthesis by acting as an electron mediator between the cathode and electrophilic substrates [39,40] as well as in reductive dehalogenations of lindane [41], chlorinated C l- hydrocarbons [42] and FREONs [114], polychlorinated biphenyl congener and hexachlorobenzene [43]. A number of bacteria are capable of reductively dehalogenating chlorinated compounds [111]. R-Cl + H++ 2e-~ R-H + C1 Particularly Methanosarcina species, which contain high concentrations of corrinoids and the Ni-factor coenzyme F430, dehalogenate chlorinated C l-hydrocarbons [112], trifluoromethane [ll3] and 1,2-dichloroethane [114] by using both coenzymes as catalyst. It was shown with M. thermophila that the corrinoid/fe-s protein of the CO-reduced carbon monoxide dehydrogenase complex was responsible for the dechlorination of trichloroethylene via dichloroethylene to ethylene [115]. The Ni/Fe-S component alone was inactive in dehalogenation. Reconstitution with the Co/Fe-S component restored the dechlorination activity, indicating that the corrinoid was the active species. The apparent K m and Vma x values were 1.7 mm TCE and 26.2 mol TCE dechlorihated min -1 (mmol factor III corrinoid)-l, comparable to the chemical dechlorination reaction catalyzed by Ti3+-reduced factor III. These findings clearly demonstrated that the Co(I) corrinoid was the active compound in enzymic dehalogenation [115]. Corrinoid-dependent O-demethylations O-Methyl ether bonds are frequently found in natural organic products and in xenobiotica. Their C-O ether bond is quite stable due to the oxygen hetero atom adjacent to the cationic center, which increases the carbo-cation stability. Ether bonds are cleaved chemically by protonolysis, since they resist oxidative or reductive OH + CH3J O--CH3 120oc Until recently it was considered that the inertness of O-methyl ethers resists bacterial degradation, particularly under anaerobic growth conditions. Bache and Pfennig, however, published in 1981 the selective isolation of the homoacetogenic Acetobacterium woodii using monomeric O-methyl ethers as carbon and energy source [116]. The bacterium, which contains high cobalamin concentrations [117], utilized the methyl groups of methoxybenzoates in the presence of carbon dioxide to release the corresponding phenols and acetic acid into the medium. Experiments with l~o-labelled methoxy aromatics proved that the oxygen remained at the nucleus [118]. This is reasonable since the O-aryl bonds are even more stable than the O-alkyl bonds. In vitro experiments with 14C-labelled methoxybenzoates and washed proteins from the homoacetogenic Sporomusa ocata strictly required Ti 3+ reduction, tetrahydrofolate and substoichiometric amounts of ATP for enzymic activity [119]. The reaction product was identified as t4clabelled CH3-tetrahydrofolate. In addition, two homomeric 14C-labelled corrinoid proteins were detected from which [lac_methyl]ch3_corrinoid has been isolated [119]. These findings are consistent with the tetrahydrofolate and ATP stimulations in vitro [120], propyl iodide inhibition [121] and [14C]acetic acid formation from radioactive methoxyl groups in vivo [122]. Nucleophilic cob(i)alamin could not substitute for tetrahydrofolate as the methyl acceptor in the O-demethylation assays [119]. However, [14Cmethyl]methyltetrahydrofolate was demethylated in a coupled enzymic reaction to tetrahydrofolate and [14C-methyl]-CH3-cobalamin. The last reac-

10 358 tion turned out to be a powerful tool in direct measurement of O-demethylations and methyltetrahydrofolate formation based on the spectral differences of cob(i)alamin and CH3-cob(III) alamin at 540 nm [119]. Remarkably, the O-demethylating enzyme system in Sporomusa ovata was non-specific towards methoxyaromatics, since more than 20 different O-methoxylated substrates with vicinal methoxyl groups were metabolized by proteins from 3,4-dimethoxybenzoategrown cells in vitro [119]. Methanol metabolism of the acetogenic bacterium Sporomusa ovata In vitro experiments with 14C-labelled substrates and growth experiments demonstrated that different corrinoid-containing methyl transferases are involved in the O-demethylations and in the methanol metabolism of S. ovata [119]. A 40-kDa corrinoid-containing protein was isolated from the cytoplasmic fraction. The homomeric protein was induced by methanol [123], and it carried a ~4Clabelled Co/3-methyl-p-cresolyl cobamide cofactor when tested with [14C]methanol in vitro [119]. Proteins from methanol-grown cells were unable to demethylate methoxybenzoates, and proteins from 3,4-dimethoxybenzoate-grown cells showed no methanol activity. This finding excludes methanol as an intermediate in O-demethylations [119]. However, the methanol system also converted methanethiol to CH3-tetrahydrofolate, a precursor of acetyl-coenzyme A. A protonation mechanism was suggested for the methanol activation [123]. The protonated methanol facilitates the nucleophilic attack on the methyl group due to the positive charge of the adjacent oxygen. This reaction parallels the mechanisms involved in transmethylations from protonated methyl-tetrahydrofolate and from S- adenosylmethionine. In addition, water is a better leaving group than OH-. CH3 CH 3 t C + S + CH 3-Tetrahydrofolate I S-Adenosylmethionine CH3 I O + H~ H Suggested methanol activation Corrinoid proteins from S. ouata contain the unique p-cresolyl cobamide which lacks a N-heterocyclic nucleotide base. Hence, p-cresolyl cobamide is 'base-off' by its chemical structure independent of its nucleotide protonation or its redox state [117]. Unlike the corrinoids in several other bacteria, p-cresolyl cobamide from S. ol,ata is a specific cofactor for enzymic activity, i.e. vitamin B12 or other corrinoids with N-heterocyclic bases could not substitute for p-cresolyl cobamide without inactivating the enzymic activities (unpublished results). EPR experiments with oxygen-inactivated ~SN-labelled corrinoid protein leaves no doubt that a histidine residue coordinated to p-cresolyl cobamide [124]. Tentative corrinoid-dependent reactions A third corrinoid-dependent reaction was proposed in eukaryotes in addition to the methionine synthase and methylmalonyl-coenzyme A mutase. A methylcobalamin-dependent 5-methylcytosine synthesis in DNA was reported [125], which would be a second pathway to DNA methylation in addition to the S-adenosylmethionine-dependent reaction. The biosynthesis of queuosine, which replaces guanosine in trnas, is another example for a corrinoid-dependent nucleotide metabolism [126]. A 2,3-epoxy precursor was reduced to queuosine under aerobic growth conditions, when cobalamin was added to the medium. Under strictly anaerobic conditions this epoxy elimination became cobalt-dependent, indicating that cobalt served as a precursor of the anaerobic de novo cobalamin biosynthesis in E. coll. Recent work identified sulfate reducers of anoxic aquatic sediments as the principal environmental methylators of mercury [127]. Since sulfate reducers demethylate methoxybenzoates [128,129], it has to be considered that a

11 359 corrinoid-dependent O-demethylation [119] occurs with a methyl transfer to positively charged metal ions. This reaction apparently takes advantage of the methyl-corrinoid properties to release the methyl not only as a carbonium ion but also as a carbanion (CH 3) [130]. The carban ion is transferred to Hg 2 on an electrophilic attack that involves a heterolytic Co-C bond cleavage: CH3-Bl2 + H20 --~ H20-Co(III ) + CH 3 CH 3 + Hg2+--~ CH3Hg + Another methyl transfer reaction involves methoxybenzoate demethylations with a subsequent formation of methylmercaptan and dimethylsulfide [131]. It might be possible that this corrinoid-dependent reaction proceeds via CH 3- tetrahydrofolate as an intermediate with an additional corrinoid-dependent methyl transfer to sulfide in a reaction apparently analogous to the methionine synthase. CH3_O. R BI2> CH3_TH F B12'9) CH3S H Bacterial corrinoid structures and human corrinoid uptake systems Only cobamides (complete corrinoids) have been obtained so far from corrinoid-containing proteins. This fact indicates that incomplete corrinoids in bacteria are presumably biosynthetic intermediates. Complete corrinoids have been isolated and analyzed by a rapid and a highly efficient HPLC method [132,117]. All corrinoid structures were then confirmed by NMR and FAB-MS spectroscopy. Another identification procedure for these corrinoids is based on the isolation and derivatization of the corrinoid nucleoside, which is subsequently analyzed using gas chromatography/mass spectrometry [133]. Cobalamin biosynthetic intermediates have been determined also by HPLC [134]. Many anaerobic bacteria produce corrinoid cofactors ( nmol (g dry cell weight)-l) which differ from vitamin B~2. Eight different corrinoids have been isolated from pure bacterial cultures; however, a particular corrinoid structure did not match a certain metabolism. Acetogenic bacteria, methanogenic bacteria, propionic acid-producing bacteria or sulfate reducers contain different corrinoids [135]. Moreover, methanogens, acetogens and Propionibacterium species were tested and were found to functionally substitute their naturally occurring corrinoid by a diverse cobamide when micromolar concentrations of an appropriate N-heterocyclic base were supplemented as a precursor in their medium [136,137]. These fermentations are known as 'guided corrinoid biosynthesis' [138]. A number of corrinoids, including imidazolyl cobalamide [139], fluorobenzimidazolyl cobamides and fluorophenolyl cobamide, have been synthesized using guided corrinoid biosynthesis. Notably, fluorinated corrinoids became physiologically active cofactors of corrinoid proteins [137]. Thus, 19F-NMR spectroscopy (Table 1) is applicable to study corrinoid-protein interactions with high sensitivity and no background in biological samples. Eukaryotes have a requirement for vitamin B12 (cobamide), although many bacteria as potential corrin producers [11,140] synthesize corrinoids Table 1 Properties of NMR nuclei Isotope Spin Natural abundance (%) Sensitivity Relative Absolute Chemical shift range (ppm) 1H 1 / C 1/ F 1/ p 1/ x x X X (30) a For endogenous phosphorus compounds.

12 360 that differ from cobalamin [135]. The daily recommended intake of 2/xg vitamin B12 in humans [141] is mediated by the corrinoid-binding proteins intrinsic factor, transcobalamin and haptocorrin [142,143]. Intrinsic factor has a preference for cobalamin. Transcobalamin accepts several complete corrinoids, and haptocorrin even binds incomplete corrinoids [139,144]. The Co-N coordination of naturally occurring cobamides was identified as an important recognition mechanism of the selective intrinsic factor-mediated uptake system in humans [139]. This specificity represents a corrinoid filter, since methylmalonyl-coa mutase activity was effected by different corrinoid cofactors [145], in contrast to the methionine synthase activity [57]. Conclusions Novel methylcorrinoid-dependent reactions have been described which form methyltetrahydrofolate, or which utilize methyltetrahydrofolate as a methyl donor. The reactive corrinoid cofactors are bound to apoproteins with different protein structures as judged from immunological and molecular biological data. A common motif of a corrinoid-binding site is not yet identified, but the protein fractions modify the corrinoid properties to improve their physiological function. It is concluded that protonated methyl donors play a central role in methyl transfers. These reactions require reductive activation by sub-stoichiometric amounts of ATP, and in most cases a CH.~ transmethylation is presumably involved after a heterolytic cleavage of the Co-CH 3 bond. References 1 Ballinger, M.D., Reed, G.H. and Frey, P.A. (1992) An organic radical in the lysine 2,3-aminomutase reaction. Biochemistry 31, Nagasawa, T., Takeuchi, K. and Yamada, H. (1988) Occurrence of a cobalt-induced and cobalt-containing nitrile hydratase in Rhodococcus rhodochrous J1. Biochem. Biophys. Res. Commun. 155, Roderick, S.L. and Matthews, B.W. (1993) Structure of the cobalt-dependent methionine aminopeptidase from Escherichia coli. A new type of proteolytic enzyme. Biochemistry 32, Rossi, M., Glusker, J.P., Randaccio, L., Summers, M.F., Toscano, P.J. and Marzilli, L.G. (1985) The structure of a BI2 coenzyme: Methylcobalamin studies by X-ray and NMR methods. J. Am. Chem. Soc. 107, Eschenmoser, A. (1988) Vitamin B12: origin of its molecular structure. Angew. Chem. Int. Ed. 27, Summers, M.F., Marzilli, L.G. and Bax, A. (1986) Complete 1H and ~3C assignment of coenzyme Bt2 through the use of new two-dimentional NMR experiments. J. Am. Chem. Soc. 108, Halpern, J. (1985) Mechanism of coenzyme Bi2-dependent rearrangements. Science 227, Stroinsky, A. (1987) Cobamide dependent enzymes. In: Comprehensive B12, Schneider, Z. and Stroinsky, A. (Eds.), Walter de Gruyter, Berlin, New York, pp Lu, W.P., Schiau, I., Cunningham, J.R. and Ragsdale, S.W. (1993) Sequence and expression of the gene encoding the corrinoid/iron-sulfur protein from Clostridium thermoaceticum and reconstitution of the recombinant protein to full activity. J. Biol. Chem. 268, Faust, L.P. and Babior, B.M. (1992) Overexpression, purification, and some properties of the AdoCbl-dependent ethanolamine ammonia-lyase from Salmonella typhimurium. Arch. Biochem. Biophys. 294, Hatanaka, H., Wang, E., Taniguchi, M., Iijima, S. and Kobayashi, T. (1988) Production of vitamin Bt2 by a fermentor with hollow-fiber module. Appl. Microbiol. Biotechnol. 27, Pett, V.B., Liebman, M.N., Murray-Rust, P, Prasad, K. and Glusker, J.P. (1987) Conformational variability of corrins: Some methods of analysis. J. Am. Chem. Soc. 109, Geno, M.K. and Halpern, J. (1987) Why does nature not use the porphyrin ligand in vitamin Bl:'? J. Am. Chem. Soc. 109, Pratt, J.M. (1993) Making and breaking the Co-alkyl bond in B12 derivatives. In: Metal lons in Biological Systems (Sigel, H. and Sigle, A, Eds.), Vol. 29, pp Marcel Dekker, New York, Basel, Hong Kong. 15 Commission on Biochemical Nomenclature (JUPAC- JUB) (1974) The nomenclature of corrinoids. Eur. J. Biochem. 45, Brown, K.L. and Hakimi, J.M. (1986) Heteronuclear NMR studies of cobalamin. 4. a-ribazole-3'-phosphate and the nucleotide loop of base-on cobalamins. J. Am. Chem. Soc. 108, Martin, B.D. and Finke, R.G. (1990) Co-C homolysis and bond dissociation energy studies of biological alkylcobalamins: methylcobalamin, including a > 1015 Co-Ctt 3 homolysis rate enhancement at 25 C following one-electron reduction. J. Am. Chem. Soc. 112, Brown, K.L. and Peck-Siler, S. (1988) Heteronuclear NMR studies of cobalamins. 9. Temperature-dependent NMR of organocobalt corrins enriched in L~C in the

13 361 organic ligand and the thermodynamics of the base-on/ base-off reaction. Inorg. Chem. 27, Hollenstein, R. and Stupperich, E. (1993) Assignment of 15N-NMR resonances of vitamin Bt2 analogues by 2D- [15N, [IH] long-range correlation: Fully [lsn] labelled Co-/3-cyano-5-hydroxybenzimidazoylcobamide (factor Ill) and derivatives. Helv. Chim. Acta, 76, Kr~iutler, B. (1987) Thermodynamic trans-effects of the nucleotide base in B12 coenzymes. Helv. Chim. Acta 70, Hogenkamp, H.P.C., Bratt, G.T. and Sun, S. (1985) Methyl transfer from methylcobalamin to thiols. A reinvestigation. Biochemistry 24, Hogenkamp, H.P.C., Bratt, G.T. and Kotchevar, A.T. (1987) Reaction of alkylcobalamins with thiols. Biochemistry 26, Kr~iutler, B. (1990) Chemistry of methylcorrinoids related to their roles in bacterial C 1 metabolism. FEMS Microbiol. Rev. 87, Chen, E. and Chance, M.R. (1990) Nanosecond transient absorption spectroscopy of coenzyme B12. Quantum yields and spectral dynamics. J. Biol. Chem. 265, R6tey, J. (1990) Enzymic reaction selectivity by negative catalysis or how do enzymes deal with highly reactive intermediates? Angew. Chem. Int. Ed. Engl. 29, Leutbecher, U., Albracht, S.P.J. and Buckel, W. (1992) Identification of a paramagentic species as an early intermediate in the coenzyme B~2-dependent glutamate mutase reaction. A cob(ii)amide? FEBS Lett. 307, Michel, C., Albracht, S.P.J. and Buckel, W. (1992) Adenosylcobalamin and cob(ii)alamin as prosthetic groups of 2-methyleneglutarate mutase from Clostridium barkeri. Eur. J Biochem. 205, Stubbe, J. (1988) Radicals in biological catalysis. Biochemitsry 27, Stubbe, J. (1989) Protein radical involvement in biological catalysis? Ann. Rev. Biochem. 58, Frey, P.A. (1990) Importance of organic radicals in enzymatic cleavage of unactivated C-H bonds. Chem. Rev. 90, Zhao, Y., Such, P. and R6tey, J. (1992) Radical intermediates in the coenzyme Bt2 dependent methyl-malonyl- CoA mutase reaction shown by ESR spectroscopy. Angew. Chem. Int. Ed. 31, Kr~iutler, B., Keller, W. and Kratkry, C. (1989) Coenzyme B~2 chemistry: The crystal and molecular structure of cob(ii)alamin. J. Am. Chem. Soc. 111, Sagi, I., Wirt, M.D., Chen, E., Frisbie, S. and Chance, M.R. (1990) Structure of an intermediate of coenzyme Bi2 catalysis by EXAFS: Cobalt(II) B12. J. Am. Chem. Soc. 112, Wirt, M.D., Sagi, I. and Chance, M.R. (1992) Formation of a square-planar Co(I)B12 intermediate. Implications for enzyme catalysis. Biophys. J. 63, Brown, K.L. (1982) Synthesis of organocobalt complexes. In: Dolphin, D. (Ed.) B12, Vol. 1, pp John Wiley & Sons, New York, NY. 36 Linn, D.E. and Gould, E.S. (1988) Electron transfer. 92. Reduction of vitamin B12 a (hydroxocobalamin) with formate and related fromyl species, lnorg. Chem. 27, Chemaly, S.M. and Pratt, J.M. (1984) The chemistry of vitamin BI2. Part 24. Evidence for hydride complexes of cobalt(ill) corrinoids. J. Chem. Soc. Dalton Trans. 1984, Lexa, D. and Saveant, J.M. (1976) Electrochemistry of vitamin B12. I. Role of the base-on/base-off reaction in the oxidoreduction mechanism of the B~2r-B12 ~ system. J. Am. Chem. Soc. 98, Scheffold, R., Abrecht, S., Orlinski, R., Ruf, H.R., Stamouli, P., Tinembart, O., Walder, L. and Weymuth, C. (1987) Vitamin Bi2-mediated electrochemical reactions in the synthesis of natural products. Pure Appl. Chem. 59, Auer, L., Weymuth, C. and Scheffold, R. (1993) Vitamin B12-catalyzed C,C-bond formation. Synthesis of a California Red Scale pheromone. Helv. Chim. Acta 76, Marks, T.S., Allpress, J.D. and Maule, A. (1989) Dehalogenation of lindane by a variety of porphyrins and corrins. Appl. Environ. Microbiol. 55, Krone, U.E., Thauer, R.K. and Hogenkamp, H.P.C. (1989) Reductive dehalogenation of chlorinated Ci-hydrocarbons mediated by corrinoids. Biochemistry 28, Krone, U.E., Thauer, P.K., Hogenkamp, H.P.C. and Steinbach, K. (1991) Reductive formation of carbon monoxide from CC[ 4 and freons 11, 12, and 13 catalyzed by corrinoids. Biochemistry 30, Assaf-Anid, N., Nies, L. and Vogel, T.M. (1992) Reductive dechlorination of a polychlorinated biphenyl congener and hexchlorobenzene by vitamin BI2. Appl. Environ. Microbiol. 58, Weissbach, H., Petrkofsky, A., Redfield, B.G. and Dickerman, H. (1963) Studies on the terminal reaction in the biosynthesis of methionine. J. Biol. Chem. 238, Pezacka, E., Green, R. and Jaboson, D.W. (1990) Glutathionylcobalamin as an intermediate in the formation of cobalamin coenzymes. Biochem. Biophys. Res. Commun. 169, Banerjee, R.V., Harder, S.R., Ragsdale, S.W. and Matthews, R.G. (1990) Mechanism of reductive activation of cobalamin-dependent methionine synthase: An electron paramagnetic resonance spectroelectrochemical study. Biochemistry 29, Banerjee, R.V., Frasca, V., Ballou, D.P. and Matthews, R.G. (1990) Participation of cob(i)alamin in the reaction catalyzed by methionine synthase from Escherichia coli: A steady-state and a rapid reaction kinetic analysis. Biochemistry 29, Gonz~iles, J.C., Banerjee, R.V., Huang, S., Summer, J.S. and Matthews, R.G. (1992) Comparison of cobalamin-independent and cobalamin-dependent methionine syn-

14 362 thase from Escherichia coli: Two solutions to the same chemical problem. Biochemistry 31, Banerjee, R.V. and Matthews, R.G. (1990) Cobalamindependent methionine synthase. FASEB J. 4, Garras, A., Djurhuus, R., Christensen, B., Lillehaug, J.R. and Ueland, P.M. (1991) A non radioactive assay for N 5-methyltetrahydrofolate-homocysteine methyltransferase (methionine synthase) based on a o-phthaldialdehyde derivatization of methionine and fluorescence detection. Anal. Biochem. 199, Frasca, V., Banerjee, R.V., Dunham, W.R., Sands, R.H. and Matthews, R.G. (1988) Cobalamin-dependent methionine synthase from E. coli B: Electron paramagnetic resonance spectra of the inactive form and the active methylated form of the enzyme. Biochemistry 27, Alston, T.A. (1991) Inhibition of vitamin Bi2-dependent microbial growth by nitrous oxide. Life Sci. 48, Haurani, F.I. (1989) The effects of free radicals on cobalamin and iron. Free Rad. Res. Commun. 7, Alston, T.A. (1991) Inhibition of vitamin Bi2-dependent methioninc biosynthesis by chloroform and carbon tetrachloride. Biochem. Pharmacol. 42, R25-R Taylor, R.T. (1982) Bi2-dependent methionine biosynthesis. In: Dolphin, D. (Ed.), B12, Vol. 2, pp John Wiley & Sons, New York, NY. 57 Kolhouse, J.F., Utley, C., Stabler, S.P. and Allen, R.H. (1991) Mechanism of conversion of human apo- to holomethionine synthase by various forms of cobalamin. J. Biol. Chem. 266, Banerjee, R.V., Johnston, N.L., Sobeski,, J.K., Datta, P. and Matthews, R.G. (1989) Cloning and sequence analysis of the Escherichia coli meth gene encoding cobalamin-dependent methionine synthase and isolation of a tryptic fragment containing the cobalamin-binding domain. J. Biol. Chem. 264, Luschinsky, C.L., Drummond, J.T., Matthews, R.G., and Ludiwig, M.L. (1992) Crystallisation and preliminary X- ray diffraction studies of the cobalamin-binding domain of methionine synthase from Escherichia coli. J. Mol. Biol. 225, Weissbach, H. and Brot, N. (1991) Regulation of methionine synthesis in Escherichia eoli. Mol. Microbiol. 5, Cai, X.-Y., Jakubowski, H., Redfiled, B., Zaleski, B., Brot, N. and Weissbach, H. (1992) Role of the metf and metj genes on the vitamin Ble regulation of methionine gene expression: Involvement of NS-methyltetrahydro - folic acid. Biochem. Biophys. Res. Commun. 182, Old, I.G., Phillips, S.E.V., Stockley, P.G. and Girons, I.S. (1991) Regulation of methionine biosynthesis in the enterobacteriaceae. Prog. Biophys. Mol. Biol. 56, Old, I.G., Margarita, D., Glass, R.E. and Girons, I.S. (1990) Nucleotide sequence of the meth gene of Escherichia coli K-12 and comparison with that of Salmonella typhimurium LT2. Gene 87, Favret, M.E. and Boeck, L.D. (1992) Effect of cobalt and cyanocobalamin on biosynthesis of A10255, a thiolpeptide antibiotic complex. J. Antibiot. 45, Simms, S.A. and Subbaramaiah, K. (1991) The kinetic mechanism of S-adenosyl-L-methionine:glutamylmethyltransferase from Salmonella typhimurium. J. Biol. Chem. 266, Warren, M.J. and Scott, A.I. (1990) Tetrapyrrole assembly and modification into the ligands of biologically functional cofactors. TIBS 15, Crouzet, J., Cameron, B., Cauchois, L., Rigault, S., Rouyez, M.C., Blanche, F., Thibaut, D. and Debussche, L. (1990) Genetic and sequence analysis of a 8.7-kilobase Pseudomonas denitrificans fragment carrying eight genes involved in transformation of precorrin-2 to cobyrinic acid. J. Bacteriol. 172, Thibaut, D., Couder, M., Crouzet, J., Debussche, L., Cameron, B. and Blanche, F. (1990) Assay and purification of S-adenosyl-L_-methionine : precorrin-2 methyltransferase from Pseudomonas denitrificans. J. Bacteriol. 172, Moss, M.L. and Frey, P.A. (1990) Activation of lysine 2,3-aminomutase by S-adenosylmethionine. J. Biol. Chem. 265, DiMarco, A.A., Bobik, T.A. and Wolfe, R.S. (1990) Unusual coenzymes of methanogenesis. Annu. Rev. Biochem. 59, Poirot, C.M., Kengen, S.W.M., Valk, E., Keltjens, J.T., van der Drift, C. and Vogles, G.D. (1987) Formation of methylcoenzyme M from formaldehyde by cell-free extracts of Methanobacterium thermoautotrophicum. Evidence for the involvement of a corrinoid-containing methyltransferase. FEMS Microbiol. Lett. 40, Van der Meijden, P., te Brommelstroet, B.W., Poirot, C.M., van der Drift, C. and Vogels, G.D. (1984) Purification and properties of methanol:5-hydroxybenzimidazolyl-cobamide methyltransferase from Methanosarcina barked. J. Bacteriol. 160, Schulz, H. and Fuchs, G. (1986) Cobamide-containing membrane protein complex in Methanobacterium. FEBS Lett. 198, Dangel, W., Schulz, H., Diekert, G., K6nig, H. and Fuchs. G. (1987) Occurrence of corrinoid-containing membrane proteins in anaerobic bacteria. Arch. Microbiol. 148, Schulz, H., Albracht, S.P.J., Coremans, J.M.C.C. and Fuchs, G. (1988) Purification and some properties of the corrinoid-containing membrane protein from Methanobacterium thermoautotrophicum. Eur. J. Biochem. 171, Thauer, R.K. (1990) Energy metabolism of methanogenic bacteria. Biochem. Biophys. Acta 1018, Blaut, M., Miiller, V. and Gottschalk, G. (1992) Energetics of methanogenesis studied in vesicular systems. J. Bioerg. Biomembr. 24, Stupperich, E., Juza, A., Eckerskorn, C. and Edelmann, L. (1990) An immunological study of corrinoid proteins

15 363 from bacteria revealed homologous antigenic determinants of a soluble corrinoid-dependent methyltransferase and corrinoid-containing membrane proteins from Methanobacterium species. Arch. Microbiol. 155, Kengen, S.W.M., Daas, P.J.H., Duits, E.F.G., Keltjens, J.T., van der Drift, C. and Vogels, G.D. (1992) Isolation of a 5-hydroxybenzimidazolyl cobamide-containing enzyme involved in the methyltetrahydromethanopterin:coenzyme M methyltransferase reaction in Methanobacterium thermoautotrophicum. Biochim. Biophys. Acta 1118, Fischer, R., G~irtner, P., Yeliseev, A. and Thauer, R.K. (1992) NS-metbyltetrahydromethanopterin:coenzyme M methyltransferase in methanogenic archaebacteria is a membrane protein. Arch. Microbiol. 158, Gfirtner, P., Ecker, A., Fischer, R., Linder, D., Fuchs, G. and Thauer, R.K. (1993) Purification and properties of NS-methyltetrahydromethanopterin : coenzyme M methyltransferase from Methanobacterium thermoautotrophicum. Eur. J. Biochem. 213, Stupperich, E., Juza, A., Hoppert, M. and Mayer, F. (1993) Cloning, sequencing and immunological characterization of the corrinoid-containing NS-methyltetrahydro - methanopterin : coenzyme M methyltransferase from Methanobacterium thermoautotrophicum. Eur. J. Biochem., in press. 83 Kaesler, B. and Sch6nheit, P. (1989) The sodium cycle in methanogenesis. CO 2 reduction to the formaldehyde level in methanogenic bacteria is driven by a primary electrochemical potential of Na + generated by formaldehyde reduction to CH 4. Eur. J. Biochem. 186, Becher, B., Miiller, V. and Gottschalk, G. (1992) The methyl-tetrahydromethanopterin :coenzyme M methyltransferase of Methanosarcina G61 is a primary sodium pump. FEMS Microbiol. Lett. 91, Becher, B., Miiller, V. and Gottschalk, G. (1992) N 5- methyl-tetrahydromethanopterin: coenzyme M methyltransferase of Methanosarcina strain G61 is a Na+-trans - locating membrane protein. J. Bacteriol. 174, Heise, R., Miiller, V. and Gottschalk, G. (1993) Acetogenesis and ATP synthesis in Acetobacterium woodii are coupled via a transmembrane primary sodium ion gradient. FEMS Microbiol. Lett. 112, Stettler, R. and Leisinger, T. (1992) Physical map of the Methanobacterium thermoautotrophicum Marburg chromosome. J. Bacteriol. 174, Van der Meijden, P., Heythuysen, H.J., Pouwels, A., Houwen, F., van der Drift, C. and Vogels, G. (1983) Methyltransferases involved in methanol conversion by Methanosarcina barkeri. Arch. Microbiol. 134, Van der Meijden, P., Heythuysen, H.J., Sliepenbeek, H.T., Houwen, F., van der Drift, C. and Vogels, G. (1983) Activation and inactivation of methanol : 2-mercaptoethanesulfonic acid methyltransferase from Methanosarcina barkeri. J. Bacteriol. 153, Van der Meijden, P., te Br6mmelstroet, B.W., Poirot, C.M., van der Drift, C. and Vogels, G. (1984) Purification and properties of methanol : 5-hydroxybenzimidazolylcobamide methyltransferase from Methanosarcina barkeri. J. Bacteriol. 160, Enssle, M., Zirngibl, C., Linder, D. and Thauer, R.K. (1991) Coenzyme F420 dependent N 5, Nl -methylen - etetrahydromethanopterin dehydrogenase in methanol grown Methanosarcina barkeri. Arch. Microbiol. 155, Van der Meijden, P., van der Lest, C., van der Drift, C. and Vogels, G. (1984) Reductive activation of methanol:5-hydroxybenzimidazolylcobamide methyltransferase of Methanosarcina barkeri. Biochem. Biophys. Res. Commun. 118, Dass, P.J.H., Gerrits, K.A.A., Keltjens, J.T., van der Drift, C. and Vogels, G.D. (1993) Involvement of an activation protein in the methanol:2-mercaptoethanesulfonic acid methyltransferase reaction in Methanosarcina barkeri. J. Bacteriol. 175, Ragsdale, S.W. (1991) Enzymology of the acetyi-coa pathway of CO z fixation. Crit. Rev. Biochem. Mol. Biol. 26, Thauer, R.K., M611er-Zinkhan, D. and Spormann, A.M. (1989) Biochemistry of acetate catabolism in an aerobic chemotropbic bacteria. Ann. Rev. Microbiol. 43, Ferry, J.G. (1992) Methane from acetate. J. Bacteriol. 174, Ragsdale, S.W., Lindahl, P.A. and Miinck, E. (1987) M6ssbauer, EPR, and optical studies of the corrinoid/ iron-sulfur protein involved in the synthesis of acetyl cenzyme A by Clostridium thermoaceticum. J. Biol. Chem. 262, Lu, W.P., Harder, S.R. and Ragsdale, S.W. (1990) Controlled potential enzymology of methyl transfer reactions involved in acetyl-coa synthesis by CO dehydrogenase and the corrinoid/iron-sulfur protein from Clostridium thermoaceticum. J. Biol. Chem. 265, Wirt, M.D., Kumar, M., Ragsdale, S.W. and Chance, M.R. (1993) X-ray absorption spectroscopy of the corrinoid/iron-sulfur protein involved in acetyl coenzyme A synthesis by Clostridium thermoautotrophicum. J. Am. Chem. Soc. 115, Harder, S.R., Lu, W.P., Feinberg, B.A. and Ragsdale, S.W. (1989) Spectroelectrochemical studies of the corrinoid/iron-sulfur protein involved in acetyl coenzyme A synthesis by Clostridium thermoaceticum. Biochemistry 28, Alber, B.E., Clements, A.P., Jablonski, P.E., Latimer, M.T. and Ferry, J.G. (1993) Enzymology of the methanogenic fermentation of acetate by Methanosarcina thermophila. In: Microbial growth on C I compounds (Murrell, J.C. and Kelly, D.P., Eds.), pp Intercept, Andover. 102 Jetten, M.S.M., Stams, A.J.M. and Zehnder, A.J.B. (1992) Methanogenesis from acetate: A comparison of the acetate metabolism in Methanothrix soehngenii and Methanosarcina spp. FEMS Microbiol. Rev. 88, Fischer, R. and Thauer, R.K. (1990) Ferredoxin-depen-

16 364 dent methane formation from acetate in cell extracts of Methanosarcina barkeri (strain MS) FEBS Lett. 269, Fischer, R. and Thauer, R.K. (1989) Methyltetrahydromethanopterin as an intermediate in methanogenesis from acetate in Methanosarcina barkeri. Arch. Microbiol. 151, Terlesky, K.C., Barber, M.J., Aceti, D.J. and Ferry, J.G. (1987) EPR properties of the Ni-Fe-C center in an enzyme complex with carbon monoxide dehydrogenase activity from acetate-grown Methanosarcina thermophila. Evidence that acetyl-coa is a physiological substrate. J. Biol. Chem. 262, Eikmanns, B. and Thauer, R.K. (1985) Evidence for the involvement and role of a corrinoid enzyme in methane formation from acetate in Methanosarcina barkeri. Arch. Microbiol. 142, Cao, X. and Krzycki, J.A. (1991) Acetate-dependent methylation of two corrinoid proteins in extracts of Methanosarcina barkeri. J. Bacteriol. 173, Grahame, D.A. (1991) Catalysis of acetyl-coa cleavage and tetrahydrosarcinapterin methylation by a carbon monoxide dehydrogenase-corrinoid enzyme complex. J. Biol. Chem. 266, Jablonski, P.E., Lu, W.P., Ragsdale, S.W. and Ferry, J.G. (1993) Characterization of the metal centers of the corrinoid/iron-sulfur component of the CO dehydrognase enzyme complex from Methanosarcina thermophila by EPR spectroscopy and spectroelectrochemistry. J. Biol. Chem. 268, Keltjens, J.T., Raemakers-Franken, C. and Vogels, G.D. (1993) Methanopterin, its structural diversity and functional uniqueness. In: Microbial Growth on C 1 compounds (Murrell, J.C. and Kelly, D.P., Eds.), pp Intercept, Andover. 111 Mohn, W.W. and Tiedje, J.M. (1992) Microbial reductive dehalogenation. Microbiol. Rev. 56, Krone, U.E., Laufer, K., Thauer, R.K. and Hogenkamp, H.P.C. (1989) Coenzyme F430 as a possible catalyst for the reductive dehalogenation of chlorinated Cl-hydrocarbons in methanogenic bacteria. Biochemistry 28, Krone, U.E. and Thauer, R.K. (1992) Dehalogenation of trifluoromethane (CFC-II) by Methanosarcina barkeri. FEMS Microbiol. Lett. 9, Holliger, C., Schraa, G., Stupperich, E., Stares, A.J.M., Zehnder, A.J.B. (1992) Evidence for the involvement of corrinoids and factor F430 in the reductive dechlorination of 1,2-dichloroethane to ethylene by Methanosarcina barkeri. J. Bacteriol. 174, Jablonski, P.E. and Ferry, J.G. (1992) Reductive dechlorination of trichloroethylene by the CO-reduced CO dehydrogenase enzyme complex from Methanosarcina thermophila. FEMS Microbiol. Lett. 96, Bache. R. and Pfennig, N. (1981) Selective isolation of Acetobacterium woodii on methoxylated aromatic acids and determination of growth yields. Arch. Microbiol. 130, Stupperich, E., Eisinger, H.J. and Kr~iutler, B. (1988) Diversity of corrinoids in acetogenic bacteria. P-cresolylcobamide from Sporomusa ot'ata, 5-methoxybenzimidazolylcobamide from Clostridium formicoaceticum and vitamin Bl2 from Acetobacterium woodii. Eur. J. Biochem. 172, DeWeerd, K.A., Saxena, A., Nagle, D.P. and Suflita, J.M. (1988) Metabolism of the l~o-methoxy substituent of 3-methoxybenzoic acid and other unlabled methoxybenzoic acids by anaerobic bacteria. Appl. Environ. Microbiol. 54, Stupperich, E. and Konle, R. (1993) Corrinoid-dependent methyl transfer reactions are involved in the methanol and in the 3,4-dimethoxybenzoate metabolism by Sporomusa ol'ata. Appl. Environ. Microbiol., in press. 120 Berman, M.H. and Frazer, A.C. (1992) Importance of tetrahydrofolate and ATP in the anaerobic O-demethylation reaction for phenylmethylethers. Appl. Environ. Microbiol. 58, Kreft, J.U. and Schink, B. (1993) Demethylation and degradation of phenylmethylethers by the sulfide-methylating homoacetogenic bacterium strain TMBS 4. Arch. Microbiol. 159, Frazer, A.C. and Young, L.Y. (1986) Anaerobic C I metabolism of the O-methyl-14C-labeled substituent of vanillate. Appl. Environ. Microbiol. 51, Stupperich, E., Aulkemeyer, P. and Eckerskorn, C. (1992) Purification and characterization of a methanol-induced cobamide-containing protein from Sporomusa ol,ata. Arch. Microbiol. 158, Stupperich, E., Eisinger, H.J. and Albracht, S.P.J. (1990) Evidence for a super-reduced cobamide as the major corrinoid fraction in vivo and a histidine residue as a cobalt ligand of the p-cresolyl cobamide in the acetogenic bacterium Sporomusa ot,ata. Eur. J. Biochem. 193, Pfohl-Leszkowicz, A., Keith, G. and Dirheimer, G. (1991) Effect of cobalamin derivatives on in vitro enzymatic DNA methylation: Methylcobalamin can act as a methyl donor. Biochemistry 30, Frey, B., McCloskey, J., Kersten, W. and Kersten H. (1988) New function of vitamin B12: cobamide-dependent reduction of epoxyqueuosine to queuosine in trnas of Escherichia coli and Salmonella typhimurium. J. Bacteriol. 170, Choi, S.C. and Bartha, R. (1993) Cobalamin-mediated mercury metbylation by Desulfoc'ibrio desulfuricans LS. Appl. Environ. Microbiol. 59, Tasaki, M., Kamagata, Y., Nakamura, K. and Mikami, E. (1992) Utilization of rnethoxylated benzoates and formation of intermediates by Desulfotomaculum thermobenzoicum in the presence or absence of sulfate. Arch. Microbiol. 157, Kuever, J., Kulmer, J., Jannsen, S., Fischer, U. and

17 365 Blotevogel, K.H. (1993) Isolation and characterization of a new spore-forming sulfate-reducing bacterium growing by complete oxidation of chatechol. Arch. Microbiol. 159, Wood, J.M. and Fanchiang, Y.T. (1979) Mechanisms for Bl~-dependent methylations. In: Vitamin B12 pp Zagalak, B and Friedrich, W., Eds.), Walter de Gruyter, Berlin, New York, NY. 131 Finster, K., King, G.M. and Bak, F. (1990) Formation of methylmercaptan and dimethylsulfide from methoxylated aromatic compounds in anoxic marine and fresh water sediments. FEMS Microbiol. Ecol. 74, Stupperich, E., Steiner, I. and RiJhlemann, M. (1986) Isolation and analysis of bacterial cobamides by High- Performance Liquid Chromatography. Anal. Biochem. 155, Sundin, D.P. and Allen, R.H. (1992) Analysis of the nucleoside moiety of cobalamin and cobalamin analogues using gas chromatography-mass spectrometry. Arch. Biochem. Biophys. 298, Blanche, F., Thibaut, D., Couder, M. and Muller, J.-C. (1990) Identification and quantification of corrinoid precursors of cobalamin from Pseudomonas denitrificans by High-Performance Liquid Chromatography. Anal. Biochem. 189, Stupperich, E., Eisinger, H.J. and Schurr, S. (1990) Corriholds in anaerobic bacteria. FEMS Microbiol. Rev. 87, Stupperich, E., Steiner, I. and Eisinger, H.J. (1987) Substitution of Coo~-(5-hydroxybenzimidazolyl)cobamide (factor III) by vitamin B12 in Methanobacterium thermoautotrophicum. J. Bacteriol. 169, Stupperich, E., Eisinger, H.J., Kerssebaum, R. and Nex0, E. (1993) Fluorinated vitamin Bl2 analogs are cofactors of corrinoid-dependent enzymes: A 19F-labeled nuclear magnetic resonance probe for identifying corrinoid-protein interactions. Appl. Environ. Microbiol. 59, Friedmann, H.C. and Thauer, R.K. (1992) Macrocyclic tetrapyrrole biosynthesis in bacteria. In: Encyclopedia of Microbiology (Lederberg, J., Ed.), Vol. 3, pp Academic Press, New York, NY. 139 Stupperich, E. and Nexo, E. (1991) Effect of the cobalt-n coordination on the cobamide recognition by the human vitamin Bt2 binding proteins intrinsic factor, transcobalamin and haptocorrin. Eur. J. Biochem. 199, Herbert, V. (1988) Vitamin Bt2: plant sources, requirements and assay. Am. J. Clin. Nutr. 48, Herbert, V. (1987) Recommended dietary intake (RDI) of vitamin B-12 in humans. Am. J. Clin. Nutr. 45, Fernandes-Costa, F. and Metz, J. (1982) Vitamin Bi2 binder (transcobalamins) in serum. Crit. Rev. Clin. Lab. Sci. 18, Gu6ant, J.L. and Nicolas, J.P (1990) Cobalamin and related binding proteins in clinical nutrition. Elsevier, Amsterdam, London, Paris. 144 Kolhouse, F. and Allen, R.H. (1977) Absorption, plasma transport, and cellular retention of cobalamin analogues in the rabbit. J. Clin. Invest. 60, Eberhard, G., Schlayer, H., Joseph, H., Fridrich, E., Utz, B. and Miiller, O. (1988) Untersuchungen zur biologischen Funktion der Nukleotidbase von Vitamin Blz. Biol. Chem. Hoppe-Seyler 369,