[26] CAROTENOID BIOSYNTHESIS (3ENE PRODUCTS 297

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1 [26] CAROTENOID BIOSYNTHESIS (3ENE PRODUCTS 297 [26] Evolutionary Conservation and Structural Similarities of Carotenoid Biosynthesis Gene Products from Photosynthetic and Nonphotosynthetic Organisms By GREGORY A. ARMSTRONG, BHUPINDER S. HUNDLE, and JOHN E. HEARST Introduction Carotenoids have attracted considerable interest as a major class of natural pigments with important roles in photosynthesis, nutrition, and photooxidative protection) The biochemical purification and characterization of carotenoid biosynthesis enzymes have proved difficult tasks, thus hindering direct structural studies of these proteins. 2 Recent developments in the molecular description of carotenoid biosynthesis genes from photosynthetic bacteria (Rhodobacter capsulatus), nonphotosynthetic bacteria (Erwinia herbicola and Erwinia uredovora), and fungi (Neurospora crassa), however, offer powerful indirect approaches for predicting structural properties of the gene products. The nucleotide sequencing of carotenoid biosynthesis genes from R. capsulatus (crta, crtb, crtc, crtd, crte, crtf, crti, crtk), 3 E. herbicol#,5 and E. uredovora (crtb, crte, crti, crtx, crty, crtz), 6 and N. crassa (al-l) 7 provides a wealth of information to be analyzed. In the absence of the purification to homogeneity of the corresponding carotenoid biosynthesis enzymes, predictions made on the basis of deduced amino acid sequences serve as a starting point for the characterization of these proteins. In this chapter we describe methods useful for the comparison and analysis of the deduced amino acid sequences of carotenoid biosynthesis enzymes from these organisms. We also discuss the significance of sequence similarities between these proteins and other gene products from t G. Britton, "The Biochemistry of Natural Pigments." Cambridge Univ. Press, Cambridge, p. D. Fraser and P. M. Bramley, this volume [33]. 3 G. A. Armstrong, M. Alberti, F. Leach, and J. E. Hearst, Mol. Gen. Genet. 216, 254 (1989). 4 G. A. Armstrong, M. Alberti, andj. E. Hearst, Proc. Natl. Acad. Sci. U.S.A. 87, 9975 (1990). 5 B. S. Hundle, M. Alberti, V. Nievelstein, P. Bcyer, G. A. Armstrong, D. Burke, H. Kleinig, and J. E. Hearst, unpublished data (1991). N. Misawa, M. Nakagawa, K. Kobayashi, S. Yamano, Y. Izawa, K. Nakamura, and K. Harashima, J. Bacteriol. 172, 6704 (1990). 7 T. J. Schmidhauser, F. R. Lauter, V. E. A. Russo, and C. Yanofsky, Mol. Cell. Biol. 10, 5064 (1990).

2 298 MOLECULAR AND CELL BIOLOGY [26] computer databases in terms of the biosynthetic reactions catalyzed and cofactor requirements. The hydrocarbon backbone of C4o carotenoids arises through a series of prenyl transfer reactions) Three molecules of the C5 building block isopentenyl pyrophosphate (IPP) are added successively to allylic substrates in 1'-4 condensations, starting with dimethylallyl pyrophosphate (DMAPP; C5), the allylic isomer of IPP. These consecutive reactions yield the products geranyl pyrophosphate (GPP; C~o), farnesyl pyrophosphate (FPP; C~5), and geranylgeranyl pyrophosphate (GGPP; C2o), which are precursors for many branches of isoprenoid metabolism. In the first reactions unique to carotenoid biosynthesis, two molecules of GGPP undergo a 1'-2-3 condensation yielding prephytoene pyrophosphate (PPPP; C4o), followed by the loss of pyrophosphate and insertion of a double bond to yield phytoene. These early reactions leading to the formation and dehydrogenation of the first C4o carotenoid phytoene are common to most carotenogenic organisms, although later biosynthetic reactions diverge? The specific biosynthetic pathways operating in R. capsulatus, ~ in Escherichia coli expressing cloned Erwinia crt genes, 6,H and in N. crassa ~2 have been summarized. Three genetic loci necessary for the synthesis of phytoene from GGPP via PPPP (crtb, crte) ~ and phytoene and dehydrogenation (crti) ~a have been identified in R. capsulatus. Cognate genes have been identified in Erwinia by comparisons of deduced sequence similarities between the R. capsulatus and Erwinia gene products and by mutational analyses. 4-6 The N. crassa crti homolog, al-1, has been identified by functional complementation and by the predicted sequence similarity of AI-I to R. capsulatus CrtI and CrtD. 7,n In vitro precursor accumulation in cell extracts from R. capsulatus mutants implicates CrtB in the condensation of two molecules of GGPP to PPPP and CrtE in the conversion of PPPP to phytoene. ~ CrtI/A 1-1 probably perform multiple dehydrogenations, converting phytoene to neurosporene in R. capsulatus (or further to lycopene and 3,4-dehydrolycopene in N. crassa), while CrtD mediates the specialized dehydrogenation of methoxyneurosporene to spheroidene observed in some photosynthetic bacteria.~2 CrtY directs the two-step con- s H. Kleinig, Annu. Rev. Plant Physiol. Plant Mol. Biol. 40, 39 (1989). 9 T. W. Goodwin, this volume [29]. io G. A. Armstrong, A. Sehmidt, G. Sandmann, and J. E. Hearst, J. Biol. Chem. 265, 8329 (1990). i~ B. S. Hundle, P. Beyer, H. Kleinig, G. Englert, and J. E. Hearst, Photochem. Photobiol. 54, 89(1991). 12 G. E. Bartley, T. J. Schmidhauser, C. Yanofsky, and P. A. Scolnik, J. Biol. Chem. 265, (1990). 13 G. Giuliano, D. Pollock, H. Stapp, and P. A. Scolnik, Mol. Gen. Genet. 213, 78 (1988).

3 [26] CAROTENOID BIOSYNTHESIS GENE PRODUCTS 299 version of the acyclic carotenoid lycopene to bicyclic fl-carotene in Erwinia.5, 6 Methods and Applications Procedures for Alignment of Deduced Amino Acid Sequences Many of the sequence comparisons described here were recognized during searches of the NBRF and SWlSS-PROT protein sequence databases using the FASTA amino acid sequence alignment program provided in the Genetics Computer Group (GCG) software package (version 6.2; June, 1990). t4 FASTA, described in detail elsewhere, 15 compares a test sequence against a databank of other sequences, ultimately yielding an alignment (opt) score in which deletions and insertions of amino acids have been considered to yield an optimal alignment. The pairwise FASTA amino acid sequence alignments were refined by maximizing the normalized alignment scores (NAS). 16 To determine the NAS, the number of identities between the two sequences are multiplied by 10 (20 in the case of cysteines). The number of gaps multiplied by 25 is subtracted from this sum. This result is divided by the average length of the two sequences and multiplied by 100, yielding the NAS. The maximal NAS method offers the advantage that sequence alignments of related proteins can be readily optimized without the use of a computer. Although the results obtained by the FASTA and NAS alignments are similar, FASTA tends to overlook regions of limited identity at the ends of aligned sequences and also yields different results in regions of low identity containing gaps. NAS refinements of the FASTA sequence alignments yielded the results shown in Figs. 1, 2, and 4. Percent identities for these pairwise comparisons, calculated using the shorter of the two sequences, are given in Table I. Sequence Alignment of CrtB, CrtE, and Other Related Protein Sequences At the time of the nucleotide sequence determination of the R. capsulatus crtb and crte genes, no similarity was observed between the deduced protein sequences and other proteins described in the literature) Sequence similarities were subsequently found between R. capsulatus CrtB and deduced protein sequences from the E. herbicola 4 and E. uredovora 6 crt genes by inspection. In addition, R. capsulatus and E. herbicola CrtB were shown, using the FASTA program, to resemble the protein product of a m4 j. Devereux, P. Haeberli, and O. Smithies, Nucleic Acids Res. 12, 387 (1984). t5 W. R. Pearson, this series, Vol. 183, p R. F. Doolittle, this series, Vol. 183, p. 99.

4 300 MOLECULAR AND CELL BIOLOGY [26] TABLE I ANALYSIS OF AMINO ACID SEQUENCE RELATIONSHIPS Sequence comparison a Number Percent Length of of Number identity b,c sequence b identities c of gaps c (%) FASTA opt ~o~ d T-pTOM5 / Eu-CrtB T-pTOM5 / Eh-CrtB T-pTOM5 / Rc-CrtB Eu-CrtB / Eh-CrtB Eu-CrtB / Rc-CrtB Eh-CrtB / Rc-CrtB Ec/IspA / Eu-CrtE Ec-IspA / Eh-CrtE Ec-IspA / Rc-CrtE Ec-IspA / Bs-GerC3 Ee-IspA / Cp-CrtE Ec-IspA / Ec-ORFX Ec-IspA / Y-HPS Eu-CrtE / Eh-CrtE Eu-CrtE / Rc-CrtE Eu-CrtE / Bs-GerC3 Eu-CrtE / Cp-CrtE Eu-CrtE / Ec-ORFX Ec-IspA / Y-HPS Eh-CrtE / Rc-CrtE Eh-CrtE / Bs-GerC3 Eh-CrtE / Cp-CrtE Eh-CrtE / EC-ORFX Eh-CrtE / Y-HPS Rc-CrtE / Bs-GerC3 Rc-CrtE / Cp-CrtE Rc-CrtE / Ec-ORFX Rc-CrtE / Y-HPS Bs-GerC3 Cp-CrtE Bs-GerC3 Ec- ORFX Bs-GerC3 Y-HPS Cp--CrtE / Ec-ORFX Cp-CrtE / Y-HPS Rc-CrtD / Eu-Crtl Rc-CrtD / Eh-Crtl Rc-CrtD / Re-Crtl Rc-CrtD / N -A1-1 Eu-Crtl / Eh-Crtl Eu-Crtl / Rc-Crtl Eu-Crtl / Ne-AI

5 [26] CAROTENOID BIOSYNTHESIS GENE PRODUCTS 301 TABLE I (continued) ANALYSIS OF AMINO ACID SEQUENCE RELATIONSHIPS Number Percent Sequence Length of of Number identity b,c FASTA comparison a sequence b identities c of gaps ~ (%) opt score d Eh-Crtl / Rc-Crtl Eh-Crtl / Nc-AI Rc-Crtl / Nc-AI a Abbreviations used here and in Fig. 1-5: T, tomato (Lycopersicon esculentum); Eu, E. uredovora; Eh, E. herbicola; Re, R. capsulatus; Ee, E. coli; Bs, B. subtilis; Cp, C. paradoxa; Y, yeast (Saccharomyces cerevisiae); Nc, N. crassa. b Using the shorter of the two compared sequences. c Alignments shown in Fig d Magnitude of the FASTA opt score increases with the length of the compared sequences at a fixed value of sequence identity. tomato edna (ptom5) (Fig. 1), 4 differentially expressed during fruit ripening. 17 Table I lists the FASTA opt alignment scores determined for pairwise comparisons of CrtB/pTOM5 sequences and shows that the identity between the ptom5 protein and E. uredovora CrtE is 25.3%. FASTA alignment values between the ptom5 protein and all three CrtB proteins are similar. Because the degree of identity in these comparisons lies close to the 25% rule-of-thumb threshold for probable homology between two sequences, t6 it is instructive to observe that many of the identities with ptom5 occur in regions most highly conserved in CrtB proteins from photosynthetic and nonphotosynthetic bacteria. Arguments for a relationship between ptom5 and carotenoid biosynthesis, such as lycopene accumulation in the ripening tomatoes in which ptom5 is differentially expressed and the similarity of the molecular weights of ptom5 and the purified bifunctional phytoene synthase from red pepper chromoplasts, ts have been detailed elsewhere. 4 The identity between CrtB proteins from photosynthetic and nonphotosynthetic bacteria is 32.7% (Table I), indicating homology between the prokary0tic sequences. In the case of CrtE, in addition to published sequence comparisons between the R. capsulatus and Erwinia proteins, 4,6 several groups have identified related protein sequences in other organisms. A gene product t7 j. Ray, C. Bird, M. Maunders, D. Grierson, and W. Schuch, Nucleic Acids Res. 15, (1987). ts B. Camara, this volume [32].

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7 [26] CAROTENOID BIOSYNTHESIS GENE PRODUCTS 303 encoded in the Cyanophora paradoxa cyanelle genome has been observed to resemble R. capsulatus CrtE and has been proposed to play an analogous role in carotenoid biosynthesis. 19 A Bacillus subtilis gene of unknown function (gerc3) 2 and a partial open reading frame (ORF) from E. coli (ORF X) 2~ were also found to exhibit sequence similarity to R. capsulatus CrtE, 2 as was the E. coli ispa gene encoding FPP synthase (FPS). 22 While preparing this chapter we also observed that yeast hexaprenyl pyrophosphate synthase (HPS) 23 displayed significant sequence similarity to CrtE and to the related proteins. These findings, summarized in the FASTA opt alignment scores (Table I) and in the refined sequence alignments (Fig. 2), are intriguing as (1) B. subtilis and E. coli do not normally synthesize carotenoids and (2) E. coli IspA, yeast HPS, and CrtE perform different isoprenoid biosynthetic reactions. For the alignments shown in Fig. 2, apart from the highly conserved Erwinia CrtE proteins (53% identity), the pairwise comparisons yield overall sequence identities of 25.6 to 32.8% (Table I), indicating probable homology) 6 CrtB and CrtE Share Domains Found in Prenyltransferases Sequences conserved between CrtE (the putative phytoene synthase) and related proteins could form domains required for the binding of related but nonidentical isoprenoid pyrophosphate substrates. The first evidence to support this theory comes from the recent observation that yeast HPS and eukaryotic FPS enzymes contain three highly conserved domains (I, II, III). 23 Domains I and II include the sequence (I,L,V)XDDXXD given in single-letter amino acid code where X can be any amino acid. These authors also observed that domain II occurs in the yeast IPP transferase, encoded by the MOD5 gene. A survey of CrtE and related proteins, using the FIND program in the GCG software package to search for core DDXXD sequences, revealed the conservation of domains I and II (Fig. 3). For E. coli IspA and the three CrtE sequences, it was necessary to allow insertion of additional amino acids in the new consensus to obtain optimal alignment in domain I (Fig. 3A; 8 of 25 residues are absolutely conserved). For domain II, residues at 6 of 13 positions are strongly conserved (Fig. 3B). Furthermore, inspection 19 C. B. Michalowski, W. L6ffelhardt, and H. J. Bohnert, J. Biol. Chem. 266, (1991). 2o D. J. Henner, EMBL database secession number M80245 (1992). 2t Y.-L. Choi, T. Nishida, M. Kawamukai, R. Utsumi, H. Sakai, and T. Komano, J. Bacteriol. 171, 5222 (1989). 22 S. Fujisaki, H. Hara, Y. Nishimura, K. Horiuchi, and T. Nishino, J. Biochem. (Tokyo) 108, 995 (1990). 23 M. N. Ashby and P. A. Edwards, J. Biol. Chem. 265, (1990).

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9 A Domain I s DD D Ec-IspA 73 VECIHAYSLIHDD Eu-CrtE 83 VEMVHAASLILDD Eh-CrtE 82 VELTHTASLMLDD Rc-CrtE 69 LELMHCASLVHDD Bs-GarC3 i01 LEMIHMASLVHDD Cp-CrtE 77 TEIIHTASLVHDD Y-RPS 182 VEMIHTASLLHDD R-FPS 92 VELLQAFFLVLDD H-FPS 92 VELLQAFFLVADD Y-FPS 89 IELLQAYFLVADD Consensus E L D D LPAMD MPCMD MPCMD LPAFD V--ID I--LD V--ID I--MD I--MD M--MD D DDDLRRG DAKLRRG NAELRRG NADIRRG DAELRRG ESDVRRG HSDTRRG SSYTRRG SSLTRRG KSITRRG RRG B Domain II s DD D Ec-IspA 215 IGLAFQVQDD ILD 227 Eu-CrtE 222 LGQAFQLLDDLTD 234 Eh-CrtE 221 FGQAFQLLDDLRD 233 Rc-CrtE 200 IGSAFQIADDLKD 212 Bs-GezC3 231 VGMSYQIIDD ILD 243 Cp-CrtE 207 LGLAFQIVDD ILD 219 Ec-ORFX ii LGTAFQLIDDLLD 23 Y-RPS 356 LGICYQLVDDMLD 368 R-FPS 235 MGEFFQIQDDYLD 247 R-FPS 235 MGEFFQIQDDYLD 247 Y-FPS 232 LGEYFQIQDDYLD 244 Y-MOD5 210 PEPLFQRLDDRVD 222 OO~OfiS~ G FQ DD D T-pTOM5 275 LGIANQLTNRLRD 287 Eu-CrtB 151 LGLAFQLTN IARD 163 Eh-CrtB 164 LGLAFQLTN IARD 176 Rc-CrtB 156 LGLAMQMSNIARD 168 FIG. 3. (A, B) CrtE, CrtB, and related proteins contain conserved domains defined in prenyltransferases. Farnesyl pyrophosphate synthase (FPS), hexaprenyl pyrophosphate synthase (HPS), and isopentenyl pyrophosphate transferase (MOD5) protein sequences not presented in Figs. I and 2 are from yeast (Y), rat (R), and human (H). The sequence used to define domains I and II (shown above in A and B; s, small or hydrophobic residue = I, L, V) was identified by aligning the FPS and HIS proteins. 23 Database accession numbers for nucleotide sequences (G, C-enBank) and protein sequences (N, NBRF) are as follows: Y-HIS, J05547(G), R-FTS, A27772(N), A34713(N), and B34713(N); H-FPS, A33415(N) and A35726(N); Y-FPS, A34441(N); Y-MOD5, A26717(N). The positions of each sequence within the respective proteins are indicated at the left and fight, with dashes indicating gaps. The new consensus shown indicates residues conserved in all 10 sequences (A) or at least 11 of 12 sequences (B). Matches with the new consensus are marked in bold type. A region similar to domain II was also found in the CrtB and ptom5 proteins (B).

10 306 MOLECULAR AND CELL BIOLOGY [26] of the CrtB (the putative PPPP synthase) and tomato ptom5 proteins revealed domain II-like sequences lacking two of the aspartate residues from the new consensus. None of the CrtE or related proteins (Fig. 2) demonstrate significant overall sequence similarity to the CrtB proteins (data not shown). Our results thus redefine the (I,L,V)XDDXXD consensus originally defined for domains I and II in eukaryotic FPS and yeast HPS enzymes, 23 to EXXXXXXLXXDDX2_+DXXXXRRG for domain I and GXXFQXXDDXXD for domain II, and suggest the occurrence of a looser domain II consensus in the CrtB and ptom5 proteins (GXXXQXXXXXXD). How can these data be rationalized in terms of the diverse biosynthetic reactions catalyzed by these enzymes? CrtB, as the putative PPPP synthase, catalyzes the 1'-2-3 condensation of two molecules of GGPP, while CrtE, as the putative phytoene synthase, binds PPPP and converts it to phytoene. The FPS and yeast HPS enzymes mediate the 1'-4 condensations ofallylic isoprenoid pyrophosphates with IPP, while yeast MOD5 donates allylic DMAPP to a trna substrate. It has been proposed that the aspartates of domains I and II form salt bridges between the FPS and HPS proteins and magnesium salts of their pyrophosphate substrates. 23 Such a hypothesis would be consistent with the participation of domains I and II in CrtE and domain II-like sequences in CrtB in isoprenoid pyrophosphate binding. The conservation of domains I and II in CrtE, in prenyltransferases (E. coli IspA, FPS, yeast HPS, MODS), and in proteins of unknown function from noncarotenogenic prokaryotes (E. coli ORFX and B. subtilis GerC3) suggests functions for the last group in biosynthetic reactions involving at least one isoprenoid pyrophosphate substrate (e.g., quinone biosynthesis, dolichol biosynthesis, trna modificationsa4). A thorough examination of the FASTA opt scores and sequences for CrtE and related proteins (Table I, Fig. 2) reveals the rough division of these sequences into two groups. The CrtE proteins and E. coli IspA form one cluster, while B. subtilis GerC3, E. coli ORFX, C. paradoxa CrtE, and yeast HPS form a second cluster. This grouping raises the possibility that C. paradoxa CrtE, proposed to be the cyanelle CrtE homolog, may rather be involved in a noncarotenogenic isoprenoid biosynthetic reaction. The eukaryotic FPS enzymes demonstrate no significant similarity outside of domains I and II to the proteins aligned in Fig. 3 (data not shown) and are hence not included in Table I. The unique nature of domains I and II was demonstrated by searching for the consensus sequences shown in Fig. 3 among all proteins in the NBRF database (release 42.0, 3/90) using the FIND program. For domain I, three searches were performed to allow for the observed spacing varia- 24 M. M. Sherman, L. A. Pe~ersen, and C. D. Poulter, J. Bacteriol. 171, 3619 (1989).

11 [26] CAROTENOID BIOSYNTHESIS GENE PRODUCTS 307 tions within the aspartate cluster. In a total of 27,711 sequences no exact matches were found excluding the proteins already described. Therefore, these motifs define fingerprints unique to proteins which bind isoprenoid pyrophosphate substrates. These examples clearly illustrate how a judicious comparison of protein sequences, always within the framework of their biological roles, can provide a starting point for suggesting enzymatic function and structural features. Sequence Alignment of Carotenoid Dehydrogenases (CrtI, CrtD, AI-1) For the carotenoid dehydrogenases, the determination of the deduced protein sequences for R. capsulatus CrtI and R. capsulatus CrtD revealed that these proteins share two highly conserved domains, one N-terminal, the other C-terminal. 3 These domains were recognized by comparing the two sequences using a homology matrix program provided in a sequence analysis software package. 25 This search was motivated by the reasoning that both proteins had been proposed to participate in analogous dehydrogenations ofphytoene for R. capsulatus CrtI and methoxyneurosporene for R. capsulatus CrtD. The predicted amino acid sequence of R. capsulatus CrtI was subsequently revised to begin at an upstream ATG codon, adding 33 new N-terminal amino acids, when it was realized that new alignments of R. capsulatus CrtI with N. crassa AI-1, ~2 with R. capsulatus CrtD, t or with E. herbicola CrtI + allowed a substantial increase in sequence similarity. Carotenoid dehydrogenase function for N. crassa A1-1 and R. capsulatus CrtI is detailed elsewhere in this volume. ~ CrtI homologs in E. herb# cola 4,5 and E. uredovora 6 were later identified by inspection of sequence similarities and by mutational analyses. FASTA sequence alignments between the carotenoid dehydrogenases are detailed in Fig. 4 and Table I. The alignments confirm the N- and C-terminal regions of sequence similarity originally noted between R. capsulatus CrtI and R. capsulatus CrtD. 3 Table I indicates that R. capsulatus CrtI, E. herbicola CrtI, and E. uredovora CrtI are closely related. Neurospora crassa A1-1 is, as the only eukaryotic carotenoid dehydrogenase, more distantly related. Rhodobacter capsulatus CrtD, as the only dehydrogenase not using phytoene as its primary substrate, is also distantly related to both the prokaryotic CrtI subgroup and to N. crassa A1-1. That the carotenoid dehydrogenases constitute an evolutionarily conserved class of proteins is revealed by the sequence identities ranging from 26.0 to 76.2% (Table I). The carotenoid dehydrogenases also show a higher degree of conservation than either the CrtB or CrtE proteins [compare identities 25 j. Pustell and F. Kafatos, Nucleic Acids Res. 10, 51 (1982). 26 G. E. Bartley, A. Kumle, P. Beyer, and P. A. Scolnik, this volume [34].

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13 [26] CAROTENOID BIOSYNTHESIS GENE PRODUCTS 309 between E. herbicola and E. uredovora for CrtI (76.2%), CrtB (64.5%), and CrtE (53.0%)]. FAD/NAD(P)-Binding Domains in Carotenoid Dehydrogenases and Cyclases Further FASTA searches comparing carotenoid dehydrogenase sequences against other proteins in the databases exposed the conservation of the N-terminal region with a variety ofeukaryotic and prokaryotic proteins known to bind FAD and/or NAD(P) cofactors 4 and with a fingerprint defined for ADP-binding folds with a/~tp structure. Similar observations were also made by other researchers. 12 Figure 5 shows the alignments of the N termini of the carotenoid dehydrogenases to each other and to the ADP-binding fingerprint. 27a8 We define a new consensus for carotenoid dehydrogenase putative ADP-binding folds on the basis of these alignments. The deduced sequences of the Erwinia CrtY (lyeopene cyclase) proteins, identified by mutational analyses, have recently become available?, 6 The two proteins are 57.6% identical, 5 including a conserved N-terminal region reminiscent of the dehydrogenase ADP-binding fold (Fig. 5). Many key residues in the dehydrogenase consensus and the fingerprint for ADPbinding folds 27a8 are conserved in the cyclases. Deviations from the fingerprint occur at the third conserved glyeine, which is replaced by asparagine, and in the size of the variable turn region between the et helix and the second/~ sheet. Interestingly, however, a revisitation of the structure of ADP-binding folds for enzymes containing both FAD and NAD(P) binding sites indicates that conservation of the third glycine in the fingerprint is not as strict as originally proposed. 29 What is the biochemical evidence for FAD/NAD(P) cofactor requirements for carotenoid dehydrogenation and lycopene cyclization? Although the N-terminal sequence alignments (Fig. 5) are suggestive, dehydrogenation and cyclization cofactor requirements have not yet been established in 27 R. K. Wierenga, P. Terpstra, and W. G. J. Hol, J. Mol. Biol. 187, 101 (1986). 28 G. Eggink, H. Engel, G. Vriend, P. Terpstra, and B. Witholt, J. Mol. Biol. 212, 135 (1990). 29 j. H. MeKie and K. T. Douglas, FEBS Lett. 279, 5 (1991). Flo. 4. Sequence alignments of carotenoid dehydrogenases. Protein sequences are labeled as in Fig. 1. The overlined region indicates a highly conserved putative ADP-binding fold for FAD/NAD(P) cofactors (see Fig. 5). Database accession numbers for nucleotide sequences (G, GenBank; E, EMBL) are as follows: Eu-CrtI, D90087(G); Eh-CrtI, M38423(G); Nc-Al-l, M33867(G); Rc-CrtD and Rc-CrtI, X52291(E).

14 310 MOLECULAR AND CELL BIOLOGY [26] ~ aaaaaaaaaaaaaa ~ bs s G G G s s s s a Eu-CztI 3 Eh-CrtI 3 Rc-CrgI I0 RC-CrtD 6 N~-AI-I 9 ConJensu~ PTVVXGAG FGGLALA IRLQAAG-- I PVLLLE QRD KPGG 38 KTVVIGAGFGGLALAIRLQAAG--IPTVLLEQRDKPGG 38 RAVVIGAGLGGLAAAMRLGAKG--YKVTVVD RLD RpGG 45 DVVVIGAEMGGLAAA IG AAAAG-- LRVTVVE AGD ApG G 41 SAIIVGAGAGGIAVAARLAKAG--VDVTVLEKNDFTGG 44 VVIGAG GGLA A RL AAG V E D PGG Eu-CrtY 6 Eh-CztM 3 DLILVGAGLANGLIALRLQQQQPDMRILLIDAAPQAGG 43 DLI L VGGG LANG L IAWRLRQ RY PQLNLLLIEAGE QPGG 40 FIG. 5. Carotenoid dehydrogenases (CrtI, CrtD, AI- 1) and yclases (CrtY) contain putative ADP-binding ]/aft folds for FAD or NAD(P) cofactors. Protein sequences are presented as in Fig. 3. Conserved N-terminal domains are compared to a fingerprint for an ADP-binding fold (shown above: G, glycine; b, basic or hydrophilic residue; s, small or hydrophobic residue; a, acidic residue; the glycines and the acidic residue are strictly conserved). 27,~ Arg-13 of Rc-CrtD (underlined) may represent the site of a point mutation as the crtd223 mutant allele of the crtd gene was sequenced. 3,4 The consensus shown indicates residues conserved in at least four of the five dehydrogenases. The gene encoding Eu-CrtY appears in the GenBank database under accession number D The sequence of the E. herbicola crty gene and the full deduced protein sequence of Eh-CrtY will be reported elsewhere. 5 prokaryotes. The daffodil chromoplast system has been used to study cofactors needed for the eukaryotic dehydrogenation and cyclization reactions, however. This work indicates a requirement for NADPH in the conversion of lycopene to fl-carotene, involving an isomerization and two cyclization reactions) 0,31 although no direct requirement for FAD/ NAD(P) cofactors was observed for the dehydrogenation. 32 In addition to the ADP-binding fold, the conservation between carotenoid dehydrogenases and FAD-binding disulfide oxidoreductases of residues involved in FAD binding and protein dimerization has been proposed. ~2~6 On the other hand, we find that additional residues involved in FAD binding in other dehydrogenases ~ are not obviously conserved in the carotenoid dehydrogenases. Future biochemical experiments to explore cofactor requirements for prokaryotic carotenoid cyclases and dehydrogenases should prove instructive. While molecular genetics cannot directly answer biochemical questions, the two fields complement each other. Specific predictions made on the basis of deduced structural features in proteins can be tested by biochemists, while biochemical properties of proteins can be dissected genetically on the molecular level. The study of carotenoid biosynthesis enzymes provides a perfect case in point. 3o p. Beyer and H. Kleinig, this series, Vol. 213 [8]. 3~ p. Beyer, U. KrOncke, and V. Nievelstein, J. Biol. Chem. 266, (1991). 32 p. Beyer, M. Mayer, and H. Kleinig, Eur. J. Biochem. 184, 141 (1989).

15 [2 7] CLONING CAROTENOID BIOSYNTHETIC GENES 31 1 Acknowledgments We thank Dr. D. J. Henner (Genentech) and Dr. H. J. Bohnert (University of Arizoua) for communicating protein sequences and sequence alignments prior to publication. This chapter is based on work supported by a National Science Foundation Graduate Fellowship (G.A.), by a National Institute of Environment and Health postdoctoral training Grant No. 2T32 ES (B.H.), by National Institutes of Health Grant GM (J.H.), and by the Office of Basic Energy Sciences, Biological Energy Division, U.S. Department of Energy, under Contract DE-ACO30-76SF00098 (J.H.). NOTE ADDED IN PROOF. Recent data indicate that CriB and CrtE homologs function in the conversion of geranylgeranyl pyrophosphate to phytoene and in the synthesis of geranylgeranyl pyrophosphate, respectively. [27] Cloning of Carotenoid Biosynthetic Genes from Maize By BRENT BUCKNER and DONALD S. ROBERTSON Introduction Several genes have been cloned from maize by a strategy commonly referred to as transposon tagging. ~-I4 For this cloning strategy, a transposon is used to induce a mutation in a gene of interest. The transposon, which should have previously been cloned, can then be used as a hybrid- l N. V. Federoff, D. B. Furtek, and O. E. Nelson, Jr., Proc. Natl. Acad. Sci. U.S.A. 81, 3825 (1984). 2 C. O'Reilly, N. S. Shepherd, A. Pereira, Z. Schwarz-Sommer, I. Bertram, D. S. Robertson, P. A. Peterson, and H. Saedler, EMBO I. 41, 877 (1985). 3 K. C. Cone, F. A. Burr, and B. Burr, Proc. Natl. Acad. Sci. U.S.A. 83, 9631 (1986). 4 U. Wienand, U. Weydemann, U. Niesbaeh-Kloesgen, P. A. Peterson, and H. Saedler, Mol. Gen. Genet. 203, 202 (1986). s M. McLaughlin and V. Walbot, Genetics 117, 771 (1987). 6 N. Theres, T. Schcele, and P. Starlinger, Mol. Gen. Genet. 209, 193 (1987). R. J. Schmidt, F. A. Burr, and B. Burr, Science 238, 960 (1987). s M. Motto, M. Maddaloni, G. Ponziani, M. Brembilla, R. Martta, N. Di Fonzo, C. Soave, R. Thompson, and F. Salamini, Mol. Gen. Genet. 212, 488 (1988). 9 S. L. Dellaporta, I. Greenblatt, J. L. Kermiele, J. B. Hicks, and S. R. Wessler, in "Chromosome Structure and Function: Impact of New Concepts, 18th Stadler Genetics Symposium" (J. P. Gustafson and R. Appels, eds.), p Plenum, New York, ~o D. R. MeCarty, C. B. Carson, P. S. Stinard, and D. S. Robertson, Plant Cell 1, 523 (1989). ~1 R. A. Martienssen, A. Barkan, M. Freeling, and W. C. Taylor, EMBO J. 8, 1633 (1989). t2 S. Hake, E. Vollbreeht, and M. Freeling, EMBO J. 8, 15 (1989). ~3 C. Leehelt, T. Pcterson, A. Laird, J. Chen, S. L. Dellaporta, E. Dennis, J. W. Peacock, and P. Starlinger, Mol. Gen. Genet. 219, 225 (1989). ~4 B. Buekner, T. U Kelson, and D. S. Robertson, Plant Cell 2, 867 (1990). METHODS IN ENZYMOLOGY, VOL. 214 ~ t 1993 by Academic Press, Inc. All rights of meprodnctlon in any form reserved.