Sequence analysis and structure prediction of type II Pseudomonas sp. USM 4–55 PHA synthase and an insight into its catalytic mechanism
© Wahab et al; licensee BioMed Central Ltd. 2006
Received: 16 June 2006
Accepted: 01 November 2006
Published: 01 November 2006
Polyhydroxyalkanoates (PHA), are biodegradable polyesters derived from many microorganisms such as the pseudomonads. These polyesters are in great demand especially in the packaging industries, the medical line as well as the paint industries. The enzyme responsible in catalyzing the formation of PHA is PHA synthase. Due to the limited structural information, its functional properties including catalysis are lacking. Therefore, this study seeks to investigate the structural properties as well as its catalytic mechanism by predicting the three-dimensional (3D) model of the Type II Pseudomonas sp. USM 4–55 PHA synthase 1 (PhaC1P.sp USM 4–55).
Sequence analysis demonstrated that PhaC1P.sp USM 4–55 lacked similarity with all known structures in databases. PSI-BLAST and HMM Superfamily analyses demonstrated that this enzyme belongs to the alpha/beta hydrolase fold family. Threading approach revealed that the most suitable template to use was the human gastric lipase (PDB ID: 1HLG). The superimposition of the predicted PhaC1P.sp USM 4–55 model with 1HLG covering 86.2% of the backbone atoms showed an RMSD of 1.15 Å. The catalytic residues comprising of Cys296, Asp451 and His479 were found to be conserved and located adjacent to each other. In addition to this, an extension to the catalytic mechanism was also proposed whereby two tetrahedral intermediates were believed to form during the PHA biosynthesis. These transition state intermediates were further postulated to be stabilized by the formation of oxyanion holes. Based on the sequence analysis and the deduced model, Ser297 was postulated to contribute to the formation of the oxyanion hole.
The 3D model of the core region of PhaC1P.sp USM 4–55 from residue 267 to residue 484 was developed using computational techniques and the locations of the catalytic residues were identified. Results from this study for the first time highlighted Ser297 potentially playing an important role in the enzyme's catalytic mechanism.
Polyhydroxyalkanoic acids (PHA) represent a complex class of biodegradable and naturally occurring biopolyesters that consist of hydroxyalkanoic acid monomers. They are produced by a wide range of bacteria as energy storage compounds especially during limited nutritional supplies and in the presence of excess carbon source. PHA synthase is the key enzyme that plays the central catalytic role in PHA production. It uses coenzymeA (CoA) thioesters of hydroxyalkanoic acids (HAs) as the main substrates and catalyzes the polymerization of HAs to yield PHA with the concomitant release of CoA [1, 2]. Numerous studies have been carried out on these enzymes and they are well characterized at the molecular level [3–5]. They can be distinguished into four types based on the subunit composition and substrate specificities [6, 7].
To date, there is no experimentally determined structural information regarding PHA synthase. However, several studies [8, 9] demonstrated that this enzyme possesses the α/β-hydrolase fold domain. The predicted three-dimensional (3D) model of Type III PHA synthase was reported using lipase as the template . For Type I and II PHA synthase enzymes, threading models had also been developed [8, 10]. Type I Ralstonia eutropha PHA synthase (PhaCRe) and Type II Pseudomonas aeruginosa PHA synthase (PhaCPa) were modeled employing the structure of lipase from Burkholderia glumae and mouse epoxide hydrolase as the templates, respectively. A lipase-box like pentapeptide motif was observed in the models and the catalytic triad was found to be located adjacent to each other [9, 10]. Further to the catalytic triad identification, another residue His453 has been identified and thought to be important in the catalytic mechanism of the enzyme in the recent type II PhaCPa model. This was confirmed by their mutagenesis studies where they found that His453 could functionally replace one of the catalytic triad's residue (His480) . Thus is it clear that with each model developed, new information was discovered regarding the structure and function of PHA synthase.
Therefore, we are motivated to perform a thorough sequence analysis of this enzyme and to predict the 3D structure of Type II PHA synthase. For this purpose, we chose PhaC1P.sp USM 4–55 as a model enzyme as this enzyme was first isolated by our group, with the ultimate aim to discover new insights on its structure and function. This is especially important in order to understand the catalytic behavior of this enzyme. Surprisingly, in our investigation of the 3D structure of PhaC1P.sp USM 4–55, an interesting feature was discovered which has never been highlighted or proposed before. We proposed an extension to the existing catalytic mechanism and that Ser297 might also be important in the formation of an oxyanion hole which might be occurring in the catalytic mechanism.
Data mining and Sequence Analysis
The linear chain of PhaC1P.sp USM 4–55 protein containing 559 residues  was subjected to various sequence analysis on SWISS-PROT , PDB  and PIR  using BLAST  and PSI-BLAST. Pair-wise and multiple sequence alignment between PHA synthase Type I, II and III were carried out using LALIGN  and CLUSTALW  respectively. Superfamily HMM  and PSI-BLAST were used to identify any conserved domains or families found in the protein.
Model Development and Evaluation
Five secondary structure prediction methods were used in this work to obtain the information on the secondary structure: PSIPRED , PHD , Prof  Sspro  and Jnet . The amino acid sequence was then threaded to the library of known folds using mGenThreader , 3DPSSM  and FUGUE . The sequence-structure alignment calculated using the threading methods was then used as the input for the development of the 3D models employing MODELLER6v2 . Optimization was carried out using DISCOVER module and MODELLER6v2. The resulted models were evaluated using PROCHECK , Verify 3D , PROVE , WHAT_CHECK  and ERRAT . The secondary structural assignment was carried out using DSSP . All the graphic presentations of the 3D model were prepared using InsightII and DSViewerPro .
Results And Discussion
Data mining and Sequence Analysis
Diverged from a common ancestor, the α/β hydrolase superfamily  of proteins is one of the largest known which includes synthases, esterases, lipases, transferases, thioesterases, haloperoxidases and many more. All of the enzymes of this family share a common fold and despite their differences in catalytic functions, these enzymes harbor a conserved catalytic amino acid sequence of the following format: nucleophile-acid-histidine, in which the nucleophile and the acid varies. The nucleophile can be cysteine, serine or aspartate, and the acid will be either aspartic acid or glutamic acid while histidine is strictly conserved. The multiple sequence alignment also showed that the conserved catalytic aspartic acid and the catalytic histidine are located at residues 451 and 479 respectively. The importance of these residues were suggested by other site-directed mutagenesis studies on the corresponding residues from other types of PHA synthases [10, 38–40]. Based on those findings as well as the sequence analysis, we propose that the catalytic residues for PhaC1P.sp USM 4–55 comprise Cys296, Asp451 and His479.
Secondary Structure Prediction
Proposed template structures obtained from three threading methods along with the calculated scores.
Human Gastric Lipase (PDB id: 1HLG)
α/β hydrolase fold
E valuea = 0.00888
Human soluble epoxide hydrolase (PDB id: 1S8O)
α/β hydrolase fold
E value = 0.327
Bromoperoxidase (PDB id: 1BRT)
α/β hydrolase fold
E value = 0.519
Haloalkane Dehalogenase (PDB id: 1BN6:A)
α/β hydrolase fold
E valueb = 3e-05
Mouse Epoxide Hydrolase (PDB id: 1CR6:A)
α/β hydrolase fold
E value = 3e-05
Human Gastric Lipase (PDB id: 1HLG:A)
α/β hydrolase fold
E value = 3e-05
Human Gastric Lipase (PDB id: 1HLG:A)
α/β hydrolase fold
Z-SCOREc = 7.86
Hydroxynitrile-Lyase (PDB id: 1QJ4)
α/β hydrolase fold
Z-SCORE = 6.31
Proline Iminopeptidase (PDB id: 1AZW)
α/β hydrolase fold
Z-SCORE = 5.08
For further assessment on the reliability of the structure-sequence alignment between 1HLG and PhaC1P.sp USM 4–55, the consensus secondary structure prediction of PhaC1P.sp USM 4–55 was used to check the alignment (Figure 4). This was achieved by comparing the aligned secondary structures of 1HLG and the consensus predicted secondary structure of PhaC1P.sp USM 4–55. Surprisingly, the structural alignment demonstrated a good alignment of 10 helices, 3 β-strands and 13 random coils altogether. The rest of the regions were shown to have a few structural mismatches and gap-containing segments that most likely will introduce errors in the predicted model.
3D Model Building and Evaluation
From the sequence analysis, it was shown that residues which are important in the catalytic activities of the enzyme reside in the α/β hydrolase fold region. Therefore, in order to elucidate the catalytic function of this protein, the 3D structure covering this region was built in this study with the structure sequence alignment between residue 267 to residue 484 of PhaC1P.sp USM 4–55 and residue 123 to residue 358 of 1HLG Chain A as shown in Figure 4. The alignment showed an expected result with the conserved catalytic residues of PhaC1P.sp USM 4–55 (Cys296, Asp451, His479) aligned with the catalytic triad of 1HLG (Ser153, Asp324, His353). It was also shown that residue Ser297 of PhaC1P.sp USM 4–55 aligned with Gln154 of 1HGL which had been agreed to contribute to the oxyanion hole formation for 1HLG . The other regions of the PhaC1P.sp USM 4–55 (residue 1 to 266; residue 485 to 559) were not modeled due to unsatisfactory alignments.
Comparison of the Cα distances for the catalytic residues between the template structure 1HGL and the predicted structure of PhaC1P.sp USM 4–55
PhaC1P.sp USM 4–55
Nucleophile – Histidine
Cys296 – His479 = 7.95 Å
Ser153 – His353 = 7.41 Å
Histidine – Acid
His479 – Asp451 = 4.74 Å
His353 – Asp324 = 4.67 Å
The resulting model of Type II PhaC1P.sp USM 4–55 consists of ten α-helices and four β-sheets which is also similar to the model of Type II PhaCPa developed by Amara and Rehm . Their model showed that the three residues thought to be important in the catalytic activities were identified to be Cys296, Asp452 and His480. This corresponds to Cys296, Asp451 and His279 identified from PhaC1 P.sp USM 4–55 3D model. Interestingly, the PhaC1P.sp USM 4–55 model revealed another important residue, Ser297, which has not been identified nor discussed before. It is proposed that this residue (highlighted in Figure 5 as well as Figure 8) contribute to the formation of the oxyanion hole and therefore is involved in the catalytic mechanism of PHA synthase. The proposed mechanism will be further discussed in the next section.
Proposed Catalytic Mechanism
To date, the catalytic mechanism of PHA synthase is still poorly understood. Based on the previous postulated catalytic mechanism initiated by both Steinbüchel and Sinskey Groups [9, 10, 43, 44] and those of Rehm and colleagues [8, 10], two subunits of PHA synthase were suggested to form a dimer when active . This dimer will then attach to the surface of the PHA granule for polymerization to take place. It was proposed that during PHA biosynthesis, the thiol group from the first subunit will act as the loading site to load the substrate 3-hydroxyacyl-CoA to the enzyme. The other thiol group from the second subunit will act as the elongation site in which it will be responsible in PHA elongation .
From the developed model, the catalytic residues for PhaC1P.sp USM 4–55 were believed to consist of Cys296 as the nucleophile, Asp451 as the conserved acid and His479 as the general base catalyst. Thus, we proposed that the catalytic mechanism for PhaC1P.sp USM 4–55 specifically and PHA synthase generally, follow the classical catalytic mechanism observed in cysteine and serine proteases  since the nature and orientation of the catalytic groups are more or less similar. From Figure 8, the distance between the imidazole group of His479 and the thiol group of Cys296 was calculated to be 3.58 Å, while the distance between the imidazole and the carboxyl group of Asp451 was 2.68 Å. These distances are indeed in the range of distance that are generally found in other α/β hydrolase fold protein such as serine and cysteine proteases as well as lipases that concerns the occurrence of the catalytic triad and the formation of the oxyanion hole [41, 46].
In contrast to the previous postulated mechanism [8, 9, 43], the above mechanism accounts for the formation of two tetrahedral intermediates (sp3 hybridization) during the PHA biosynthesis. These transition state intermediates have been widely agreed to be present in all cysteine proteases and lipases [45, 47, 48] but have not been suggested before in PHA biosynthesis. A vital point to consider here is whether or not this enzyme contains another active site known as the oxyanion hole. This is an important conserved feature of the α/β hydrolase fold protein that is present to stabilize the tetrahedral intermediate state. For the enzyme to work effectively, this unstable transition state needs some form of stabilization. An oxyanion hole is said to occur during the stabilization of the highly negatively charged oxyanion (O-) of the tetrahedral intermediate by two hydrogen bonds from the surrounding amide (NH) group such that the NH groups are pointing towards the oxyanion [37, 45, 47, 49]. Several studies have demonstrated that the presence of the oxyanion hole is significant in stabilizing the tetrahedral intermediate in various proteins [46, 50, 51]. For α/β hydrolase fold proteins, the mandatory feature of the occurrence of the oxyanion hole is that one of the residues that is involved in the hydrogen bonding with the oxyanion is always located next to the nucleophile (Ser297 in PhaC1P.sp USM 4–55). From the 3D model, it is very clear that Ser297 could contribute its NH group to stabilize the negatively charge oxyanion formed by the enzyme-substrate complex due to its close proximity to the nucleophile. The orientation of this residue can be observed in Figure 7. Our proposed role of Ser297 in the stabilization of the tetrahedral intermediate could explain why serine is conserved with almost all of the Type II PHA synthase. However, the second NH group that is involved in the stabilization of the tetrahedral intermediate in PhaC1P.sp USM 4–55 could not be identified due to the lack of information on the orientation of the enzyme-substrate complex. Perhaps knowledge of how the enzyme interacts with the substrate would probably shed some light into identifying the second residue that contributes to the formation of the oxyanion hole if this feature is indeed present in the PHA synthase catalytic mechanism.
We have performed sequence analysis and attempted to predict the 3D structure of PhaC1P.sp USM 4–55 using the method of fold recognition due to very low similarity to any available experimentally solved 3D protein structures. A series of molecular modeling and computational methods were combined in order to gain insight into the 3D structure of PhaC1P.sp USM 4–55 concentrating on the α/β hydrolase fold region. Human gastric lipase was used as the modeling template and from the developed model, it was shown that the catalytic residues were located adjacent to each other. From the sequence analysis and the deduced model, we have also identified Ser297, which we proposed to be involved in the catalytic activity by forming an oxyanion hole. This finding has never been proposed by any other studies before. We also proposed an extension to the catalytic mechanism of this enzyme based on that of serine and cysteine proteases. Two tetrahedral intermediates were postulated to occur during the PHA biosynthesis and we believe that the formation of an oxyanion hole is integral to the catalytic mechanism of this enzyme.
This work is supported by the Top Down Grant No 09-02-04-001 BTK/TD/004 awarded by the National Biotechnology Directorate, Ministry of Science, Technology and Innovation, Malaysia. The authors wish to acknowledge The National Biotechnology and Bioinformatics Network (NBBNet) for providing the computing resources in our laboratory.
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