Analysis of membrane stereochemistry with homology modeling of sn-glycerol-1-phosphate dehydrogenase

Hiromi Daiyasu1,2, Takaaki Hiroike1, Yosuke Koga3 and Hiroyuki Toh1

1 Department of Computational Biology, Biomolecular Engineering Research Institute, 6–2–3, Furuedai, Suita, Osaka 565-0874 and 3 Department of Chemistry, School of Medicine, University of Occupational and Environmental Health, Yahatanishi-ku, Kitakyushu, Fukuoka 807-8555, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Different enantiomeric isomers, sn-glycerol-1-phosphate and sn-glycerol-3-phosphate, are used as the glycerophosphate backbones of phospholipids in the cellular membranes of Archaea and the remaining two kingdoms, respectively. In Archaea, sn-glycerol-1-phosphate dehydrogenase is involved in the generation of sn-glycerol-1-phosphate, while sn-glycerol-3-phosphate dehydrogenase synthesizes the enantiomer in Eukarya and Bacteria. The coordinates of sn-glycerol-3-phosphate dehydrogenase are available, although neither the tertiary structure nor the reaction mechanism of sn-glycerol-1-phosphate dehydrogenase is known. Database searching revealed that the archaeal enzyme shows sequence similarity to glycerol dehydrogenase, dehydroquinate synthase and alcohol dehydrogenase IV. The glycerol dehydrogenase, with coordinates that are available today, is closely related to the archaeal enzyme. Using the structure of glycerol dehydrogenase as the template, we built a model structure of the Methanothermobacter thermautotrophicus sn-glycerol-1-phosphate dehydrogenase, which could explain the chirality of the product. Based on the model structure, we determined the following: (1) the enzyme requires a Zn2+ ion for its activity; (2) the enzyme selectively uses the pro-R hydrogen of the NAD(P)H; (3) the putative active site and the reaction mechanism were predicted; and (4) the archaeal enzyme does not share its evolutionary origin with sn-glycerol-3-phosphate dehydrogenase.

Keywords: Archaea/chirality/glycerol phosphate dehydrogenase/membrane


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Since the first report about Archaea by Woese and Fox (Woese and Fox, 1977Go), many studies related to the distinctions among the three superkingdoms of life have been made. One of the most striking characteristics that distinguish Archaea from other organisms is the cellular membrane structure. Each organism synthesizes glycerophosphates as the backbones of glycerophospholipids, which are a major component of the biological membrane. The stereochemistry of the glycerophosphate of Archaea is enantiomeric with those of Eukarya and Bacteria. In Archaea, the glycerophosphate exists as sn-glycerol-1-phosphate (G1P), whereas Eukarya and Bacteria use sn-glycerol-3-phosphate (G3P) (Kates, 1978Go). Both glycerophosphates are generated from a common substrate, dihydroxyacetonephosphate (DHAP) and the chirality of each product is determined by the enzyme of the organism (Figure 1Go). G1P is produced by sn-glycerol-1-phosphate dehydrogenase (G1PDH) in Archaea, while the formation of G3P is catalyzed by sn-glycerol-3-phosphate dehydrogenase (G3PDH) in Eukarya and Bacteria. Analyses of such natural protein engineering would provide great insights for the design of enzymes to generate products with different chirality.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 1. Biosynthesis of phospholipids in different organisms.

 
The genes encoding G1PDH and the putative orthologues have been found only within Archaea. Recent genome analyses have revealed that one copy of G1PDH or the putative orthologous gene is present within each archaeal genome. However, the biochemical characterizations of G1PDH have been performed with only two enzymes, derived from Methanothermobacter thermautotrophicus (Nishihara and Koga, 1997Go) and Aeropyrum pernix K1 (Han et al., 2002Go). The studies revealed that G1PDH is an NAD(P)H-dependent enzyme and that the enzyme can produce G1P selectively. Koga et al.(1998)Go showed that G1PDH has amino acid sequence similarity to glycerol dehydrogenase (GDH) and alcohol dehydrogenase type IV (ALDH). On the other hand, G1PDH does not show significant sequence similarity to G3PDH. Based on these observations, they concluded that G1PDH and G3PDH have different evolutionary origins, in spite of the similarity in both the substrate and product.

The coordinates of G3PDH are available (Suresh et al., 2000Go), although the crystal structure lacks the substrate. In contrast, the structure of G1PDH has not yet been determined. Therefore, the differences between G1PDH and G3PDH that are related to the production of their specific enantiomers remain unclear. To investigate this problem, we tried to predict the tertiary structure of G1PDH. First, the relatives of G1PDH with coordinates available in the PDB were searched with PSI-BLAST. We found 81 sequences that showed significant sequence similarity to G1PDH. Of the detected sequences, the tertiary structures of two enzymes, GDH and dehydroquinate synthase (DHQS), are available. GDH showed relatively higher similarity to G1PDH than DHQS. Therefore, a model structure of M.thermautotrophicus G1PDH was built by homology modeling from the crystal structure of GDH. The molecular mechanism for the chirality of the product will be discussed based on the model structure and the coordinates of G3PDH.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Sequence and structural data

The sequence data with significant similarity to G1PDH were collected by database searching with PSI-BLAST (Altschul et al., 1997Go) at the NCBI site (http://www.ncbi.nlm.nih.gov/blast/psiblast.cgi). The database searching was performed against the nr-db. The amino acid sequence of G1PDH from M.thermautotrophicus was used as a query for the search. The ID codes and the corresponding sequence databases for G1PDH-related proteins are shown in Figure 3Go. The amino acid sequences of the G3PDH relatives were also collected by database searching with the G3PDH sequence from Bacillus subtilis.




View larger version (131K):
[in this window]
[in a new window]
 
Fig. 3. An unrooted NJ tree of G1PDH and its relatives. The sequence is indicated by the source name, the sequence database (sp, SwissProt; pir, PIR; gb, GenBank; pdb, PDB) and the ID code. The sequences derived from Archaea are colored blue and those from Eukarya are colored red. The bootstrap probability of the clustering at a node is indicated, when the node is critical for the evolutionary discussion.

 
The data from two structures, GDH derived from Bacillus stearothermophilus (pdb: 1JQ5) and DHQS from Emericella nidulans (pdb: 1DQS), were used for a structural alignment. The coordinates of GDH were also used as the template for building a model structure of M.thermautotrophicus G1PDH.

Sequence and structural alignments

A preliminary analysis with the collected sequences of G1PDH and its relatives revealed that they could be roughly classified into four closely related groups. First, a multiple alignment of each group was constructed with the alignment software CLUSTAL W 1.74 (Thompson et al., 1994Go). Then, the four multiple alignments were piled up using the profile alignment function in CLUSTAL W. The alignment thus obtained was slightly modified by visual inspection, to keep the gaps from interrupting the secondary structure elements as much as possible. A structural alignment between GDH and DHQS was used as the reference for the operation. The structural alignment was organized from a program based on the double dynamic programming algorithm (Taylor and Orengo, 1989Go). In this analysis, a modified program by one of the authors was used (Toh, 1997Go; Daiyasu and Toh, 2000Go).

Secondary structural prediction

The secondary structure of G1PDH derived from M.thermautotrophicus was predicted with the PHDsec program at the EMBL PredictProtein server (http://www.embl-heidelberg.de/predictprotein) (Rost and Sander, 1993Go, 1994Go; Rost, 1996Go), for comparison with those of the known structures of GDH and DHQS.

Phylogenetic analysis

To investigate the evolutionary relationships between G1PDH and its relatives, a molecular phylogenetic tree was constructed by the neighbor-joining (NJ) method (Saitou and Nei, 1987Go). For the analysis, the sites including gaps were excluded from the multiple alignment.

The genetic distance between every pair of aligned sequences was calculated as the maximum likehood (ML) estimate (Felsenstein, 1996Go), using the JTT model (Jones et al., 1992Go) for the amino acid substitutions. Based on these distances, an NJ tree was constructed for all of the sequences included in the alignment. The statistical significance of the NJ tree topology was evaluated by a bootstrap analysis (Felsenstein, 1985Go) with 1000 iterative tree constructions.

For the phylogenetic analysis, two software packages, PHYLIP 3.5c (Felsenstein, 1993Go) and MOLPHY 2.3b3 (Adachi and Hasegawa, 1996Go), were used. The tree was drawn by TreeView (Page, 1996Go).

Homology modeling

Three crystal structures of the GDH from B.stearothermophilus have been reported, with pdb codes 1JQ5, 1JQA and 1JPU (Ruzheinikov et al., 2001Go). 1JQ5 includes all of the coordinates of the amino acid residues and those of the Zn2+ ions and the NAD+. The two remaining pdb data entries lack some coordinates of the protein, whereas 1JQA includes the Zn2+ ions and the substrate, glycerol. Since these crystal structures are very similar to each other, the structure of 1JQA was superimposed on that of 1JQ5. Based on the superimposition, the coordinates of glycerol were introduced into the 1JQ5 data. Thus, the coordinates of 1JQ5, including the glycerol data, were used as the template for the G1PDH model building.

To make the model structure, first the amino acid residues of 1JQ5 were substituted with the corresponding residues of G1PDH derived from M.thermautotrophicus, according to the multiple sequence alignment. At this stage, neither insertions nor deletions were introduced into the model. Next, the glycerol in the model was replaced with DHAP, a substrate for G1PDH (see Results and discussion for details). Then, the side chains of the model structure were subjected to energy minimization under conditions in which the main chains and the included substances were restrained by a harmonic function. After the operation, the insertions and deletions were introduced into the model structure, according to the alignment. The regions modified by the insertions or deletions, together with two surrounding residues from both sides, were subjected to the energy minimization. Finally, the energy minimization was executed for all of the atoms of the model structure, including the Zn2+ ion, the NAD+ and the DHAP.

The biopolymer module of InsightII Ver. 2000 (Molecular Simulations Inc.) was used for the introduction of amino acid substitutions, insertions and deletions into the model structure. PRESTO (Morikami et al., 1992Go) Ver. 3, a protein simulation tool, was used for the energy minimization. Throughout the modeling procedure, the energy minimization was performed by the conjugate gradient method with an AMBER C99 force field (Cornell et al., 1995Go; Wang et al., 2000Go) and the calculation was applied to the model in the vacuum. The dielectric constant was set to 1.0. The zinc ion in the model was treated as a point charge. A distance cut-off method was adopted for the calculation of the electrostatic interaction (cut-off distance = 15.0 Å).


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Phylogenetic relationships of G1PDH relatives

After two iterations of PSI-BLAST using M.thermautotrophicus G1PDH as a query, 82 sequences with E-values <=0.005 were detected. Of these, 65 sequences were used in the following study, because 17 sequences showed high sequence divergence to the query G1PDH. The 65 sequences included two proteins with crystal structures that have been determined. One of them is GDH from B.stearothermophilus (pdb: 1JQ5) (Ruzheinikov et al., 2001Go) and the other is DHQS from E.nidulans (pdb: 1DQS) (Carpenter et al., 1998Go). Both structures are classified into the same superfamily in the structure classification database, SCOP (Murzin et al., 1995Go) (http://scop.mrc-lmb.cam.ac.uk/scop). The r.m.s.d. for the superimposition of 213 C{alpha} atoms between GDH and DHQS is 1.6 Å, in spite of their low sequence identity (14%) (Ruzheinikov et al., 2001Go). The sequence identity between M.thermautotrophicus G1PDH and 1JQ5 is also weak (21%). No sequence similarity to G3PDH was detected in this database search.

The 65 proteins were roughly classified into four groups according to their sequence similarities and enzymatic functions. A multiple alignment of these sequences was constructed. To save space, the alignment of 22 sequences, which were selected as representatives of the four groups, is shown in Figure 2Go. The actual secondary structures of GDH and DHQS and the predicted secondary structure of M.thermautotrophicus G1PDH are also shown in Figure 2Go. Based on the alignment of the 65 sequences, a molecular phylogenetic tree was constructed by the NJ method (Figure 3Go). The tree was divided into four clusters rooted at nodes A, B, C and D, which correspond to the four groups described above.



View larger version (103K):
[in this window]
[in a new window]
 
Fig. 2. A multiple alignment of G1PDH and its relatives. A number in parentheses indicates the length of the sequence omitted from the figure. The gap for an insertion and/or deletion is indicated by a hyphen. A measure indicating the site number is shown over the alignment. Arrows indicate the alignment sites, which are discussed in the text. The number neighboring each arrow is the residue number of M.thermautotrophicus G1PDH. When physicochemically similar residues occupy more that 80% of the total of the 65 sequences at a site, the residues are indicated by inverse character. The amino acid residues are classified into six physicochemically similar groups, based on the criteria of Dayhoff et al. (Dayhoff et al., 1978Go): (1) hydrophobic group, L, I, M and V; (2) aromatic group, F, Y and W; (3) small hydrophilic group, S, T, P, A and G; (4) negatively charged group, D, E, Q and N; (5) positively charged group, K, R and H; and (6) Cys residue, C. The putative Zn2+ ion binding sites are shown in pink. The predicted {alpha}-helices and ß-strands of M.thermautotrophicus G1PDH are indicated by red and blue lines, respectively. Likewise, those in the crystal structures derived from GDH and DHQS are indicated by bars with the corresponding color. The region from alignment sites 3 to 393 was used for the homology modeling.

 
The first cluster rooted at node A consisted of the 15 sequences of G1PDH and the putative orthologues derived from Archaea and the seven sequences derived from Bacteria. The bootstrap probability for the clustering at node A was 97.7%. Since every archaeal genome available today encodes either one G1PDH or a putative orthologue, the protein is considered to be essential for Archaea. The G1PDH and the putative orthologues from Archaea were rooted at node E. The bootstrap probability at node E was 86.3%. Since neither the structure nor the function of the seven bacterial ORF products is known, the 15 archaeal sequences were used as the G1PDH group for the following study. As shown in Figure 2Go, the predicted secondary structures of M.thermautotrophicus G1PDH corresponded to the actual secondary structures of 1JQ5 and 1DQS very well.

The second cluster rooted at node B comprised nine sequences of bacterial GDH and its relatives. The bootstrap probability for the clustering at node B was 100.0% and this cluster is referred to as the GDH group. GDH catalyzes the oxidation of glycerol to dihydroxyacetone in the glycerolipid metabolism pathway (May and Sloan, 1981Go). Glycerol, the substrate of GDH, shares the same carbon frame with DHAP, the substrate of G1PDH, although the phosphate group does not exist in glycerol. GDH shows stereospecificity for the pro-R hydrogen (HR) at C4 on the nicotinamide ring of NAD(P)H and requires a Zn2+ ion for its activity (You, 1985Go; Mallinder et al., 1992Go). This group included one sequence from fission yeast and one archaeal ORF product derived from Halobacterium sp. NRC-1 (gb: AE005158). According to the sequence similarity, AE005158 is annotated as G1PDH in the genome analysis of Halobacterium sp. NRC-1 (Ng et al., 2002). However, EGSA_HALN1, which is also derived from Halobacterium sp. NRC-1 and belongs to the G1PDH group in Figure 3Go, is also annotated as G1PDH. The tree topology shown in Figure 3Go suggests that AE005158 belongs to the GDH group, rather than the G1PDH group. Considering that no counterpart of AE005158 has been detected in any other archaeal genome, the gene for AE005158 may have been introduced into the genome of Halobacterium sp. NRC-1 from Bacteria by horizontal gene transfer.

The 14 sequences of DHQS and its relatives constituted the third cluster, rooted at node C. The bootstrap probability for the clustering was 100.0% and this cluster is referred to as the DHQS group. In addition to the previously reported sequences derived from Bacteria, fungi and plants, the five ORF products derived from Archaea were detected as members of this group. The function of DHQS is the conversion of 3-deoxy-D-arabino-heptulosonate-7-phosphate to dehydroquinate in the second step of the shikimate pathway, which is required for the synthesis of aromatic amino acids (Bentley, 1990Go). The catalytic mechanism of DHQS consists of complex, multi-step reactions (Srinivasan et al., 1963Go). The first step of the reactions in DHQS is a hydrogen transfer between the hydroxyl group of the substrate and NAD(P)+, which is similar to the reaction catalyzed by G1PDH and GDH. Like GDH, the Zn2+ ion is required for the activity of DHQS (Lambert et al., 1985Go).

The fourth cluster consisted of 20 iron-containing alcohol dehydrogenases. The bootstrap probability for the clustering at node D was 99.3%. The cluster showed low sequence identities to the other three clusters and was distinguished from the other clusters. The cluster is referred to as the ALDH group and was used as the outgroup of the other three clusters. The substrate specificity varied even within the ALDH group. Several members are known to require the Zn2+ ion(s) for their activities (de Vries et al., 1992Go). This group included 10 ORF products derived from Archaea, with actual functions that have not yet been determined.

This study, together with the previous reports (Koga et al., 1998Go), suggests that the G1PDH group shares a common ancestry with the GDH, DHQS and ALDH groups. The members of the four groups are able to catalyze the hydrogen transfer, although each group is involved in different metabolic pathways. In spite of the statistical significance of the clustering at node D, the relationship among the G1PDH, GDH and DHQS groups was obscure. That is, the G1PDH and GDH groups formed a cluster at node F with a bootstrap probability of only 30.7%, before the DHQS group is connected to them. The alignment was also subjected to a molecular phylogenetic analysis by the ML method (Felsenstein, 1981Go). Since the number of sequences used for the NJ tree was too large for the ML analysis, representative sequences were selected from each group. The relationship of the four groups in the ML tree was the same as that of the NJ tree (data not shown).

The relatively close relationship between the G1PDH and GDH groups was suggested by both the NJ and the ML trees, although no statistical significance for the clustering of the two groups was shown by either tree. Furthermore, the number of gaps between the G1PDH and GDH groups was less than that between the G1PDH and DHQS groups (see Figure 2Go). Based on these observations, together with the similarity in the carbon frames of the substrates of G1PDH and GDH, the structure of GDH was adopted as the template for the model building of the G1PDH structure in this study.

Amino acid residues conserved in the G1PDH group

The multiple alignment shows that the residues involved in NAD+ binding in the crystal structures of GDH and DHQS are strongly conserved over the four groups (alignment sites 114, 115, 119, 141, 149 and 190 in Figure 2Go). In addition, the residues involved in Zn2+ ion binding, which have been identified in the crystal structures of GDH and DHQS, were also strongly conserved in all of the groups (alignment sites 200, 293 and 314 in Figure 2Go). The observation is consistent with the NAD(P)H requirement of G1PDH (Nishihara and Koga, 1997Go; Han et al., 2002Go), although the involvement of a Zn2+ ion in the activity of G1PDH has not been reported. The alignment suggested that G1PDH requires Zn2+ ion(s) for the reaction and that Asp168, His248 and His264 of the M.thermautotrophicus protein (alignment sites 200, 293 and 314 in Figure 2Go) are involved in Zn2+ ion binding. In the crystal structure of GDH, two Asp residues, at alignment sites 148 and 200, form hydrogen bonds with the hydroxyl oxygen atoms of the substrate, respectively. As described above, alignment site 200 was conserved among the four groups, while alignment site 148 was conserved within the family, except for the ALDH group. The conservation of the residues in the G1PDH group suggests that the corresponding residues of G1PDH are involved in the interaction with the oxygen atoms of DHAP. Three positively charged residues of the DHQS group, at the alignment sites 154, 297 and 380, are considered to be involved in the interaction with the phosphate of the substrate (Carpenter et al., 1998Go). Two of them (alignment sites 297 and 380) were also strongly conserved and occupied by positively charged residues in the G1PDH group, although the sites were not conserved in the GDH group. The absence of the phosphate in the substrate of GDH would explain this observation.

In the X-ray analysis of GDH, the structure was separated into two domains by a deep cleft (Ruzheinikov et al., 2001Go). The N-terminal {alpha}/ß domain constitutes one side of the cleft and is involved in the NAD+ binding. This domain shows structural similarity to the Rossmann fold, although it deviates somewhat from the classical Rossmann fold, such as in the inverted orientation to NAD+. The N-terminal domain corresponded to the alignment sites 1–189 in Figure 2Go. Many amino acid residues in the domain were conserved among the four groups, as described above. The C-terminal domain of GDH is further separated into two subdomains. One of the subdomains corresponded to the region of alignment sites 190–280, which forms the floor of the active site of GDH. The other subdomain, which corresponded to the region after alignment site 280, forms the other side of the cleft and is involved in the substrate binding. The sequence divergence between every pair of groups was remarkable in the C-terminal domain, which may reflect their substrate specificity. Within the G1PDH group, however, the C-terminal domain was conserved, as well as the N-terminal domain.

Homology modeling

As described above, we adopted the coordinates of the crystal structure of B.stearothermophilus GDH as the template for homology modeling. The requirement of a Zn2+ ion for G1PDH has not been reported. However, we introduced a Zn2+ ion for model building of G1PDH, because of the requirement of the Zn2+ ion for the catalytic activities of GDH, DHQS and ALDH and the conservation of the amino acid residues involved in the Zn2+ ion binding. One of the possible roles of the Zn2+ ion is that it may polarize a target hydroxyl group of the substrate to facilitate the hydride transfer and proton loss (Carpenter et al., 1998Go).

The structure of DHAP was constructed based on the carbon frame of the glycerol molecule in the template structure. Although the reduced form of the substrate is G1P, which corresponds to glycerol in GDH, the discussion of DHAP is essential because of the reversible reaction in G1PDH. As the first step, the hydroxyl group at C2 of the glycerol was replaced with the carbonyl oxygen, which can accept a hydrogen from NAD(P)H. Next, we replaced one of the hydroxyl groups of the intermediate model of the substrate with a phosphate group. The selection was determined as follows. By analogy with the reaction mechanism suggested for GDH (Ruzheinikov et al., 2001Go), the oxygen atoms bound to two contiguous carbons were considered to contribute to the interaction with the Zn2+ ion in G1PDH. One of the oxygen atoms is derived from the replaced carbonyl oxygen. Another oxygen comes from a hydroxyl group at either end of the substrate. The selection was inevitably determined from the distance from the Zn2+ ion. The hydrogen group in question could not be replaced with a phosphate group, because there was no room for such a bulky group, owing to the many amino acid residues crowded within the space. Therefore, the remaining hydroxyl group at the carbon furthest from the Zn2+ ion was replaced with the phosphate.

The model structure of M.thermautotrophicus G1PDH complexed with the substrate is shown in Figure 4aGo. The detailed structure near the putative active site is shown in Figure 4bGo. Like the tertiary structure of GDH, G1PDH consists of two domains separated by a deep cleft. The NADH, the Zn2+ ion and the DHAP are present in the cleft. The locations of the residues conserved over the four groups can be roughly classified into three spatial clusters, the N-terminal Rossman-fold like domain, the vicinity of the metal ion and the inside of the cleft (data not shown). Asp168, His248 and His264 of M.thermautotrophicus G1PDH (alignment sites 200, 293 and 314 in Figure 2Go) coordinate with the Zn2+ ion in the model structure. Asp121 and Asp168 (alignment sites 148 and 200 in Figure 2Go) form hydrogen bonds with the carbonyl oxygen at C2 of DHAP, while the corresponding residues in GDH form hydrogen bonds with a hydroxyl group at one terminus of the glycerol.




View larger version (82K):
[in this window]
[in a new window]
 
Fig. 4. Model structure of M.thermautotrophicus G1PDH. (a) {alpha}-Helices and ß-strands are indicated by orange cylinders and yellow arrows, respectively. (b) The putative active site of the model structure. The inset shows the predicted catalytic mechanism for G1PDH.

 
As described above, three amino acid residues of DHQS, Lys152, His275 and Lys356 (at the alignment sites 154, 297 and 380, respectively), are considered to form a phosphate-binding pocket for its substrate (Carpenter et al., 1998Go). The three residues correspond to Arg127, His252 and Arg320 of the M.thermautotrophicus G1PDH. In the model structure, hydrogen bonds exist between Arg127, His252 and the oxygen atoms of the phosphate group of DHAP. Arg320 of G1PDH did not interact with the phosphate of the substrate and was found on the surface of the model structure. In DHQS, the corresponding residue (Lys356) also seemed to function as the dimer interface, in addition to the phosphate-binding pocket. This observation suggests that Arg320 may at least function as the interface in G1PDH. Since glycerol is not used as the substrate for G1PDH (Nishihara and Koga, 1997Go), the phosphate group of the DHAP is considered to be important for the molecular recognition by G1PDH.

The reaction model of G1PDH and the stereochemistry of the product

The model structure thus obtained can explain the generation of an enantiomeric product by G1PDH. If the DHAP is located at the putative active site of the model structure, as shown in Figure 4Go, then HR at C4 on the NADH nicotinamide ring can attack the C2 carbon of the substrate in the presence of the Zn2+ ion. The location of the hydrogen atom of NADH determines the stereochemistry of the product of G1PDH. The location of HR described above is suited to the generation of G1P, instead of G3P. The distance between the C2 of DHAP and the HR in the model structure is 2.3 Å, which is short enough for the reaction. The reaction model is also shown, in Figure 4bGo.

We determined the location of the substrate in the model structure, based on the location of the glycerol in GDH. However, three other possible locations of DHAP against the nicotinamide ring of NAD(P)H were considered. We examined the four locations of DHAP, in order to check whether the DHAP location in the model could be uniquely determined. The four patterns of the DHAP locations are shown in Figure 5Go. Pattern I corresponds to the model structure shown in Figure 4Go. Patterns I and II indicate the HR selective reaction. GDH from B.stearothermophilus catalyzes the HR transfer to the substrate (Spencer et al., 1989Go). The presence of the substrate and the Zn2+ ion on the same side as HR in the GDH structure is consistent with the hydrogen stereospecificity for the cofactor (Ruzheinikov et al., 2001Go). The crystal structure of DHQS complexed with the inhibitor suggests the possibility of HR selectivity of the cofactor (Carpenter et al., 1998Go). G1P is generated by the location of the substrate in pattern I, while the location of the substrate in pattern II facilitates the production of G3P. Therefore, the location of the substrate in pattern II can be ignored. In both patterns III and IV, the substrate is located on the same side as HS. As described above, however, the catalytic reactions are HR selective in the homologues, GDH and DHQS. The conservation of the residues in G1PDH, which are involved in Zn2+ binding in GDH and DHQS, suggests that the Zn2+ ion is present on the HR side of G1PDH. Therefore, the substrate could not interact with the Zn2+ ion in the location within patterns III and IV. In addition, there seems to be no room around the HS side in the model structure for the substrate to interact, because of the presence of the N-terminal Rossmann-fold like domain on this side, as in the cases of GDH and DHQS. Hence neither pattern III nor IV location was suitable for the reaction by G1PDH. Therefore, only the location within pattern I remained as a model to explain the chirality of the product. That is, the model structure of G1PDH supported the stereochemistry of the reaction mechanism for the archaeal enzyme.



View larger version (9K):
[in this window]
[in a new window]
 
Fig. 5. The possible locations of DHAP against NAD(P)H.

 
Comparison of catalysis between G3PDH and G1PDH

We also searched for G3PDH and its relatives through the amino acid sequence database, using the B.subtilis sequence of G3PDH as a query. As a result, 114 sequences including the orthologues of G3PDH showed significant sequence similarity to the query. Of the sequences thus obtained, 31 sequences showed similarity to only the N-terminal 100 residues of G3PDH, which included pyrroline-5-carboxylate reductase, 3-hydroxyacyl-CoA dehydrogenase, ketol acid reductoisomerase (acetohydroxy acid isomeroreductase), UDP-glucose 6-dehydrogenase and octopine dehydrogenase. The remaining 250 residues of G3PDH showed significant sequence similarity only to G3PDH and its putative orthologues (data not shown). G1PDH and its relatives were not detected by this database search. The detected sequences included only one protein with available coordinates, the G3PDH derived from Leishmania mexicana (Suresh et al., 2000Go). The crystal structure of G3PDH consists of two domains, the N-terminal and the C-terminal domains. The N-terminal domain, composed of about 190 amino acids, is involved in the NAD binding, while the substrate-binding activity is performed by the C-terminal domain. The C-terminal domain is rich in {alpha}-helices and shows structural similarity to ketol acid reductoisomerase, although there is no significant sequence similarity between them. In spite of the domain constitution similarity, that is, the N-terminal Rossmann-fold like domain followed by the C-terminal helical domain, neither GDH nor DHQS shows significant tertiary structure similarity to G3PDH. Furthermore, the Rossmann-fold like domain of G3PDH is categorized into a different fold to those of GDH and DHQS in SCOP (Murzin et al., 1995Go). The dissimilarity in tertiary structure and amino acid sequence suggests different evolutionary origins of G1PDH and G3PDH, although their catalytic functions are similar to each other.

The details of the G3PDH reaction mechanism have not been established, although the HS specificity and the absence of an essential metal ion for the activity have been reported (Edgar and Bell, 1980Go; Suresh et al., 2000Go). In addition, proteins such as 3-hydroxyacyl-CoA dehydrogenase, ketol acid reductoisomerase, UDP-glucose 6-dehydrogenase and octopine dehydrogenase, which were detected as relatives of G3PDH by the database search, are also known to show HS specificity (You, 1985Go). The difference in the hydrogen stereospecificity also suggests the different evolutionary origins between G1PDH and G3PDH.

The model structure of G1PDH is compared with the crystal structure of G3PDH in Figure 6Go, where the nicotinamide rings of the NADH cofactors from both structures are superimposed. As described above, the DHAP, the Zn2+ ion and the putative active site within the G1PDH model exist on the same side as HR. Suresh et al.(2000)Go indicated that Lys125, Lys210–Asp211, Asp263, Thr267 and Arg274–Asn275 constitute the active site of the L.mexicana G3PDH according to the conservation of the residues, although the substrate was not included in the crystal structure. These residues are present on the same side as HS in the G3PDH structure. That is, the putative active site of G3PDH is present on the opposite side from that of G1PDH, across the nicotinamide ring. As in the case of G1PDH, we could consider the four possible locations of the substrate against the NAD(P)H ring in G3PDH. In this case, patterns I and II in Figure 5Go were excluded, owing to the HS selectivity of G3PDH. Pattern IV was also rejected, because the product derived from the orientation was G1P. Therefore, pattern III remained as the model for the location of the substrate in G3PDH. That is, the DHAP in G3PDH is considered to occupy a quasi-symmetric position against the plane of the nicotinamide ring, relative to that in G1PDH.



View larger version (96K):
[in this window]
[in a new window]
 
Fig. 6. Comparison of the active sites between G1PDH and G3PDH. The nicotinamide ring of NADH in the model structure of G1PDH is superimposed with that in the crystal structure of G3PDH. The predicted active site of G1PDH is shown in a green oval and that of G3PDH in a pink oval.

 
Conclusion

We have discussed the origin of chirality in the membrane constituents between Archaea and other organisms, based on the homology modeling of G1PDH. The comparison between G1PDH and G3PDH is expected to provide deep insights into functional convergence and protein engineering. Similar products, except for the chirality, are generated from different enzymes. Both enzymes have the Rossmann-fold like domain, although they are considered to have different origins [see the discussions about DHQS (Carpenter et al., 1998Go) and GDH (Ruzheinikov et al., 2001Go)]. That is, the Rossmann-fold like domains were generated by convergent evolution. Therefore, analyses of G1PDH and G3PDH would be a great source of information not only about the evolution of living organisms in the ancient stage, but also about the selection of an enzyme designed to produce an enantiomer.


    Notes
 
2 To whom correspondence should be addressed. E-mail: daiyasu{at}kuicr.kyoto-u.ac.jp Back


    Acknowledgments
 
This research was partly supported by a research grant endorsed by the New Energy and Industrial Technology Development Organization (NEDO).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Adachi,J. and Hasegawa,M. (1996) MOLPHY (Programs for Molecular Phylogenetics), 2.3b3. Institute of Statistical Mathematics, Tokyo.

Altschul,S.F., Madden,T.L., Schaffer,A.A., Zhang,J., Zhang,Z., Miller,W. and Lipman,D.J. (1997) Nucleic Acids Res., 25, 3389–3402.[Abstract/Free Full Text]

Bentley,R. (1990) Crit. Rev. Biochem. Mol. Biol., 25, 307–384.[ISI][Medline]

Carpenter,E.P., Hawkins,A.R., Frost,J.W. and Brown,K.A. (1998) Nature, 394, 299–302.[CrossRef][ISI][Medline]

Cornell,W.D., Cieplak,P., Bayly,C.I., Gould,I.R., Merz,K.M.,Jr, Ferguson,D.M., Spellmeyer, D.C., Fox,T., Caldwell,J.W. and Kollman,P.A. (1995) J. Am. Chem. Soc., 117, 5179–5197.[ISI]

Daiyasu,H. and Toh,H. (2000) J. Mol. Evol., 51, 433–445.[ISI][Medline]

Dayhoff,M.O., Schwartz,R.M. and Orcutt,B.C. (1978) In Dayhoff,M.O. (ed.), Atlas of Protein Sequence and Structure. National Biomedical Research Foundation, Washington, DC, pp. 345–358.

de Vries,G.E., Arfman,N., Terpstra.P. and Dijkhuizen.L. (1992) J. Bacteriol., 174, 5346–5353.[Abstract]

Edgar,J.R. and Bell,R.M. (1980) J. Biol. Chem., 255, 3492–3497.[Free Full Text]

Felsenstein,J. (1981) J. Mol. Evol., 17, 368–376.[ISI][Medline]

Felsenstein,J. (1985) Evolution, 39, 783–791.[ISI]

Felsenstein,J. (1993) PHYLIP (Phylogeny Inference Package), Version 3.5c. Department of Genetics, University of Washington, Seattle, WA.

Felsenstein,J. (1996) Methods Enzymol., 266, 418–427.[ISI][Medline]

Han,J.S., Kosugi,Y., Ishida,H. and Ishikawa,K. (2002) Eur. J. Biochem., 269, 969–976.[Abstract/Free Full Text]

Jones,D.T., Taylor,W.R. and Thornton,J.M. (1992) Comput. Appl. Biosci., 8, 275–282.[Abstract]

Kates, M. (1978) Prog. Chem. Fats Other Lipids, 15, 301–342.[Medline]

Koga,Y., Kyuragi,T., Nishihara,M. and Sone,N. (1998) J. Mol. Evol., 46, 54–63.[ISI][Medline]

Lambert,J.M., Boocock,M.R. and Coggins,J.R. (1985) Biochem. J., 226, 817–829.[ISI][Medline]

Mallinder,P.R., Pritchard,A. and Moir,A. (1992) Gene, 110, 9–16.[CrossRef][ISI][Medline]

May,J.W. and Sloan,J. (1981) J. Gen. Microbiol., 123, 183–185[ISI]

Morikami,K., Nakai,T., Kidera,A., Saito,M. and Nakamura,H. (1992) Comput. Chem., 16, 243–248.[CrossRef][ISI]

Murzin,A.G., Brenner,S.E., Hubbard,T. and Chothia,C. (1995) J. Mol. Biol., 247, 536–540.[CrossRef][ISI][Medline]

Ng,W.V. et al. (2000) Proc. Natl Acad. Sci. USA, 97, 12176–12181.[Abstract/Free Full Text]

Nishihara,M. and Koga,Y. (1997) J. Biochem., 122, 572–576.[Abstract]

Page,R.D. (1996) Comput. Appl. Biosci., 12, 357–358.[Medline]

Rost,B. (1996) Methods Enzymol., 266, 525–539.[CrossRef][ISI][Medline]

Rost,B. and Sander,C. (1993) J. Mol. Biol., 232, 584–599.[CrossRef][ISI][Medline]

Rost,B. and Sander,C. (1994) Proteins, 19, 55–72.[ISI][Medline]

Ruzheinikov,S.N., Burke,J., Sedelnikova,S., Baker,P.J., Taylor,R., Bullough,P.A., Muir,N.M., Gore,M.G. and Rice,D.W. (2001) Structure (Camb.), 9, 789–802.[CrossRef][ISI][Medline]

Saitou,N. and Nei,M. (1987) Mol. Biol. Evol., 4, 406–425.[Abstract]

Spencer,P., Bown,K.J., Scawen,M.D., Atkinson,T. and Gore,M.G. (1989) Biochim. Biophys. Acta, 994, 270–279.[ISI][Medline]

Srinivasan,P.R., Rothchild,J. and Sprinson,D.B. (1963) J. Biol. Chem., 238, 3176–3182.[Free Full Text]

Suresh,S., Turley,S., Opperdoes,F.R., Michels,P.A. and Hol,W.G. (2000) Struct. Fold. Des., 8, 541–552.[ISI][Medline]

Taylor,W.R. and Orengo,C.A. (1989) J. Mol. Biol., 208, 1–22.[ISI][Medline]

Thompson,J.D., Higgins,D.G. and Gibson,T.J. (1994) Nucleic Acids Res., 22, 4673–4680.[Abstract]

Toh,H. (1997) Comput. Appl. Biosci., 13, 387–396.[Abstract]

Wang,J., Cieplak,P. and Kollman,P.A. (2000) J. Comput. Chem., 21, 1049–1074.[CrossRef][ISI]

Woese,C.R. and Fox,G.E. (1977) Proc. Natl Acad. Sci. USA, 74, 5088–5090.[Abstract]

You,K.S. (1985) CRC Crit. Rev. Biochem., 17, 313–451.[ISI][Medline]

Received August 1, 2002; revised August 30, 2002; accepted September 2, 2002.





This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (6)
Request Permissions
Google Scholar
Articles by Daiyasu, H.
Articles by Toh, H.
PubMed
PubMed Citation
Articles by Daiyasu, H.
Articles by Toh, H.