Detailed description of overall and active site architecture of PPDC- 3dThDP, PPDC-2HE3dThDP, PPDC-3dThDP-PPA and PPDC- 3dThDP-POVA

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1 Online Supplemental Results Detailed description of overall and active site architecture of PPDC- 3dThDP, PPDC-2HE3dThDP, PPDC-3dThDP-PPA and PPDC- 3dThDP-POVA Structure solution and overall architecture of PPDC-3dThDP and PPDC-2HE3dThDP Previously a structure of phenylpyruvate decarboxylase of Azospirillum brasilense (AbPPDC) in complex with the cofactors ThDP and Mg 2+ was solved to a resolution of 1.5 Å (denominated PPDC-ThDP) (1). Binary complexes of AbPPDC with 3deazaThDP (PPDC-3dThDP) and 2-(1- hydroxyethyl)3deazathdp (PPDC-2HE3dThDP) were obtained by incubating AbPPDC, that was purified without addition of ThDP, with an excess of both compounds respectively. After two days of incubation the complete exchange of ThDP with 3dThDP or 2HE3dThDP was confirmed by the total loss of enzyme activity. Crystal structures of PPDC-3dThDP and PPDC-2HE3dThDP were solved to 3.2 and 1.85 Å resolution, using molecular replacement with a subunit of PPDC- ThDP (PDB 2NXW) as a search model. In both structures, clear electron density was present for the cofactor/intermediate analogues in the two active sites of the homodimer in the asymmetric unit (a.u.), and for most of the protein backbone. However, neither the residues of the N-terminal his-tag, nor the last few C-terminal residues were included in the model due to a lack of electron density. Also, in both binary complexes a large peptide region in the vicinity of the active sites, spanning residues 104 to120, could only be modeled partially due to very poorly defined electron density. The asymmetric unit of the crystal structures contains a homodimer, where each subunit adopts the archetypical pyruvate oxidase (POX) fold consisting of three distinct α/β-type domains designated PYR, R and PP domain (1). Each dimer contains two equivalent active sites which are shared between the PYR domain of one subunit and the PP domain of the neighboring subunit. Very little changes in subunit architecture are observed in PPDC-3dThDP and PPDC- 2HE3dThDP compared to PPDC-ThDP (see Supplemental Table 1S online for a list of rms deviations). The biological tetramers (2) of PPDC-3dThDP and PPDC-2HE3dThDP can be obtained from the homodimers in the a.u. through the two-fold crystallographic symmetry axis. However, the nonperpendicular arrangement of non-crystallographic axes relating the monomers in the a.u. and the crystallographic axis results in an asymmetrical tetramer assembly, best described as an asymmetrical dimer of dimers (see Figure 5a and Supplemental Table 2S online for interface surfaces). A same quaternary arrangement was also found for PPDC-ThDP (See Supplemental Table 1S online) (1). Structure solution and overall architecture of PPDC-3dThDP-PPA and PPDC-3dThDP- POVA The catalytically inert binary PPDC-3dThDP complex was subsequently used for cocrystallization with the substrates phenylpyruvic acid (PPA) and 5-phenyl-2-oxo-valeric acid (POVA). Therefore the pre-incubated PPDC-3dThDP complex was incubated for a few hours 1

2 with the substrates before crystallization setups were started. Crystal structures of the PPDC- 3dThDP-PPA and PPDC-3dThDP-POVA ternary complexes were solved to 2.15 and 1.9 Å resolution using the same molecular replacement approach as above. In both structures clear electron density was present for cofactor analogues and substrate molecules in both active sites in the a.u. Although clear electron density was now present for the loop , density was missing for the N-terminal his-tag, the C-terminal residues and a region spanning residues 331 to 340 connecting the R and PP domains. In the two ternary complexes clear electron density corresponding to an additional substrate molecule was also present in a site remote from the active site. This regulatory site is present in every subunit at the juncture of the PYR, R and PP domains, lined by residues Arg60, Arg214, Arg215, Met238, Arg240, Leu395, Met396 and Ala397. In the asymmetric units of the two ternary complexes two subunits, related by a noncrystallographic two-fold axis, form a homodimer. Relatively large differences occur in the overall geometry of the subunits of the ternary complexes (PPDC-3dThDP-PPA and PPDC- 3dThDP-POVA) compared to the binary complexes (PPDC-ThDP, PPDC-3dThDP and PPDC- 2HE3dThDP) as exemplified by large rms- deviations upon superposition (Supplemental Table 1S online). Subunits of PPDC-3dThDP-PPA and PPDC-3dThDP-POVA on the other hand superimpose nearly perfectly. The difference in subunit geometry between the ternary and binary complexes is mainly caused by a difference in domain orientation. When the PYR and PP domains are superimposed, the R domains differ by a rotation of about 12 (Figure 4). This domain rotation is accompanied by the closing of the active sites in the ternary complexes via a large rearrangement of active site loop of the R-domain and the concomitant ordering of the active site loop of the PYR domain of the neighboring subunit. The rearrangement of both active site loops is clearly coupled since they interact with each other in the closed active site conformation. The reorganization of the two active site loops finally leads to a slight tilting of the C-terminal helix which closes further over the active site pocket. Similar to the tetramers of the binary complexes (PPDC-ThDP, PPDC-3dThDP and PPDC- 2HE3dThDP), the tetramers of the tertiary complexes (PPDC-3dThDP-PPA and PPDC-3dThDP- POVA) can be obtained from two dimers related by a two-fold crystallographic symmetry axis. There are however large differences in the assembly of the tetramers (Figure 5). For the ternary complexes, the non-crystallographic symmetry axes relating the two subunits in the dimer intersect with the crystallographic axis at an angle of 90, resulting in a dimer of dimers with pseudo 222 symmetry. In going from the asymmetrical dimer of dimers observed for the binary complexes to the symmetrical dimer of dimers of the ternary complexes, one dimer has to be rotated by about 34, vis-à-vis to the second dimer. This also has implications for the dimer-dimer interfaces. While the AC and BD interfaces are different in the asymmetrical tetramers (see Figure 5, for subunit nomenclature) these interfaces are the same for the symmetrical tetramers. Comparable to the BD interface in the asymmetrical tetramer, the AC and BD dimer-dimer interfaces in the symmetrical tetramers are formed by an extension of the six-stranded β-sheet of the R domains to the adjacent R-domains. Folding and reorganization of the active site loops, where loop of one subunit interacts with loop of the adjacent subunit, cause an increase in the monomer-monomer interface area in the ternary complexes (see Supplemental Table 2S online). On the other hand, the reorganization of the asymmetrical tetramer into a symmetrical tetramer upon substrate binding in the active and regulatory site is not accompanied by a significant change in dimer-dimer interface area. Active sites of the binary PPDC-3dThDP and PPDC-2HE3dThDP complexes 2

3 Despite the relatively low resolution of the PPDC-3dThDP structure, the 3dThDP molecules could be unambiguously modeled with full occupancy in the electron density in both active sites of the homodimer in the a.u. (Figure 2a). Upon superposition of the PPDC-3dThDP and PPDC- ThDP structures the cofactors and active site residues superimpose nearly perfectly (see (1) for a detailed description of the active site architecture of PPDC-ThDP). The 3dThDP molecules adopt the typical V conformation, with Φ T = and Φ P = (compare to ThDP in PPDC- ThDP: Φ T = and Φ P = ). The PPDC-2HE3dThDP structure shows clear electron density in the two active sites of the a.u. for both the 3deazaThDP moiety and the 1-hydroyethyl moiety of the intermediate analogue (Figure 2b). The density for the 1-hydroyethyl moiety however can be best interpreted as a mixture of the R and S enantiomers at the Cα atom, consistent with the fact that a racemic mixture was used in the co-crystallization. Consequently both enantiomers were modeled with half occupancy. Slight deviations of the dihedral angles typical for the V conformation of the cofactor are observed relative to ThDP or 3dThDP caused by the covalent addition of the hydroxyethyl moiey (Φ T = and Φ P = for the S enantiomer, and Φ T = and Φ P = for the R enantiomer). The electron density does not a priori allow to distinguish between the methyl and hydroxyl groups attached to the Cα. However, for both enantiomers the hydroxyethyl moieties were modeled with good confidence with their hydroxyl group pointing toward the 4 amino group of the aminopyrimidine ring of 3dThDP based on a distance of 2.5 Å between the N4 and the hydroxyl group. In the R configuration the methyl group is pointing toward the large hydrophobic cavity lined by Met380, Phe385, Met461, Phe465 and Phe532. This configuration thus allows straightforward modeling of the phenyl group of the physiological intermediate 2-(1-hydroxy-2-phenyl-ethyl)-ThDP in the active site (Figure 7a). The S enantiomer does not allow modeling of the phenyl moiety without generating extensive clashes with protein atoms. Therefore we conclude that the R enantiomer is the relevant species, which is used for further interpretation. Except for a small rotation of the side chain of Phe465 and Asp25 (in one active site only) no changes in side chain conformation occur in the active site of PPDC-2HE3dThDP compared to PPDC-ThDP or PPDC-3dThDP. No direct interactions are formed between any amino acid residue and the hydroxyethyl moiety. The C2α-hydroxyl group is located 2.5 Å from the cofactor s N4 amino/imino group, in an ideal orientation to form a strong interaction. Finally we find an enzyme-bound water molecule located at 3.5 Å from the C2α. This water in its turn is hydrogen bonded to the Asp25 carboxylate group and to the side chain hydroxyl of Thr71. Active sites of the ternary complexes PPDC-3dThDP-PPA and PPDC-3dThDP-POVA The PPDC-3dThDP-PPA and PPDC-3dThDP-POVA structures shows the respective substrates, phenylpyruvic acid and 5-phenyl-2-oxo-valeric acid, bound with full occupancy in the two active sites of the homodimer in the asymmetric unit (Figure 2c and d). No significant difference in cofactor analogue geometry is observed compared to PPDC-3dThDP or PPDC-ThDP (Φ T = and Φ P = for PPDC-3dThDP-PPA and Φ T = and Φ P = for PPDC-3dThDP-POVA). Substrate binding leads to a reorganization of the active site loops spanning residues 104 to 120 and 280 to 294 respectively. Folding of the loop over the reaction pocket positions two additional active site residues, His112 and His113. In this process a hydrogen bond is formed between His112 and Asp282 of the loop of the neighboring subunit. The reorganization of the second active site loop also permits some new interactions with the C-terminal helix (e.g. between the side chain of Gln536 and Arg538 and the main chain carbonyls of Asp282 and of Ala 287 and Ser288 respectively) allowing this helix to close further over the active site. 3

4 The keto-acid moieties of PAA and POVA are bound in a similar way in the active site of PPDC (Figure 2). The C2α atoms of the substrates are located about 3.4 Å from the C2 of the 3dThDP, poised for nucleophilic attack by the cofactor. The 2-keto group is located within interaction distance of His113 and the N4 of the cofactor s aminopyrimidine ring. The carboxyl oxygens are hydrogen bonded to the main chain amide of Asp25, the side chain amine of His113 and to a water molecule which is located at an extremely short distance of about 2.3 Å from the carboxyl oxygen. This water molecule is also hydrogen bonded to the side chains of Thr71 and Asp25. Asp25 also forms a hydrogen bond with His112 of the first active site loop, which in turn is hydrogen bonded to Asp282 of the second active site loop. Since Asp282 is exposed to solvent the triad of ionizable residues Asp25-His112-Asp282 could serve as a proton relay shuttling protons into the active site. Leu462 is wedged directly underneath the carboxyl group of the substrate. Interestingly this leucine is not conserved in most other ThDP-dependent decarboxylases such as PDC s and IPDC s, where it is replaced by a glutamate residue (1;2). In the latter enzymes a catalytic role has been proposed for this glutamate (3;4). The methylene groups bridging the keto-acid and aromatic moiety of the substrate are bound in a hydrophobic region lined by residues Met380, Phe385, Ala402, Met461 and Phe465 (Figure 2c and d). The phenyl group of both substrates is stacked on one face in a parallel fashion to the side chain of His112, and on the other face in an anti-parallel fashion to the side chain of Phe532. This latter interaction seems more ideal for POVA than for PPA, which might account for the higher affinity of AbPPDC for POVA (2). The outer part of the active site is mainly lined by residues Asp282 and Thr283 from the loop, and several residues from the C-terminal helix: Phe532, Gln536 and His540. The latter residues move closer over the active site when the smaller substrate PAA is bound, thus suggesting that the C-terminal helix acts as a modular switch which can adapt according to the size of the side group of the substrate. Comparison of the substrate activation mechanism of PPDC to the proposed mechanism for S. cerevisiae PDC The signal transduction pathway observed here for PPDC differs from the most commonly accepted mechanism for substrate activation in S. cerevisiae PDC. In the latter enzyme, a cysteine (Cys221) is considered as the site of covalent binding of the regulatory substrate molecule. This binding information is then proposed to be transmitted via His92, Glu91 and Trp412 to the ThDP cofactor (5;6). Since Cys221 is not conserved in PPDC the same mechanism cannot apply for this enzyme (1). The PPDC regulatory site is situated at a distance of 9 Å from the residue corresponding to Cys221 (Glu212). Although the regulatory binding site in PPDC is different from the proposed site in PDC, both signal transduction routes have in common that the residues involved in this route are located at the interface of the PYR, R and PP domains (see Supplemental Figure1 online). Possibly a signal transduction route has evolved independently (or divergently) in PDC and PPDC. On the other hand, the regulatory substrate binding pocket of PPDC seems to a certain extent conserved in PDC. The two arginine residues which form an interaction with the carboxyl of the substrate in PPDC are conserved in PDC (Arg63 and Arg224). Arg214 which forms a cation-π stacking with the aromatic moiety of the substrate in PPDC is replaced by a serine (Ser223). These residues surround a cavity which might harbor a pyruvate molecule. However, the entrance to this cavity is obstructed by the side chain of Tyr405, but a simple change in side chain rotamer could open up this site for substrate entry. The possible role of this site in PDC remains to be investigated. 4

5 References 1. Versées, W., Spaepen, S., Vanderleyden, J., and Steyaert, J. (2007) FEBS J. 274, Spaepen, S., Versées, W., Gocke, D., Pohl, M., Steyaert, J., and Vanderleyden, J. (2007) J.Bacteriol. In Press 3. Schütz, A., Golbik, R., König, S., Hübner, G., and Tittmann, K. (2005) Biochemistry 44, Sergienko, E. A. and Jordan, F. (2001) Biochemistry 40, Wang, J., Golbik, R., Seliger, B., Spinka, M., Tittmann, K., Hübner, G., and Jordan, F. (2001) Biochemistry 40, Baburina, I., Li, H., Bennion, B., Furey, W., and Jordan, F. (1998) Biochemistry 37,

6 Figure legends: Supplemental Figure 1: Comparison of the substrate activation mechanisms of PPDC and S. cerevisiae PDC. A superposition in ribbon representation is shown of PPDC-3dThDP- POVA in dark grey and S. cerevisiae PDC (PDB 1QPB) in light grey. The residues relevant to the activation mechanism in PDC, Cys221, His92, Glu91 and Trp412, are shown as grey sticks. The corresponding residues in PPDC are shown as green and cyan sticks depending on the subunit they belong to. The cofactor analogue is shown in yellow and the substrates in the active and regulatory sites of PPDC in magenta. While both activation mechanisms are clearly different and use a different initial trigger, they both use the interface of the PYR, R and PP domains to propagate the signal from the regulatory site to the active site. 6

7 Supplemental Table 1S. Comparison of PPDC structures: Rms-deviation (in Å) upon superposition (510 Cα atoms) PPDC- 3dThDP PPDC- 2HE3dThDP PPDC- 3dThDP- PPA PPDC-ThDP Monomer-monomer Dimer-dimer Tetramer-tetramer PPDC-3dThDP Monomer-monomer Dimer-dimer Tetramer-tetramer PPDC-2HE3dThDP Monomer-monomer Dimer-dimer Tetramer-tetramer PPDC-3dThDP-PPA Monomer-monomer 0.25 Dimer-dimer 0.29 Tetramer-tetramer 0.32 PPDC- 3dThDP- POVA 7

8 Supplemental Table 2S. Tetramer architecture: accessible surface area buried in the subunit interfaces (in Å 2 ) Monomer-monomer interface Dimer-dimer interface PPDC-ThDP a PPDC-3dThDP PPDC-2HE3dThDP PPDC-3dThDP-PPA PPDC-3dThDP- POVA a Data from 1 8

9 Supplemental Figure 1 9