Lipases provide a new mechanistic model for polyhydroxybutyrate (PHB) synthases: characterization of the functional residues in Chromatium vinosum PHB synthase

Polyhydroxybutyrate (PHB) synthases catalyze the conversion of beta-hydroxybutyryl coenzyme A (HBCoA) to PHB. These enzymes require an active site cysteine nucleophile for covalent catalysis. A protein BLASTp search using the Class III Chromatium vinosum synthase sequence reveals high homology to prokaryotic lipases whose crystal structures are known. The homology is very convincing in the alpha-beta-elbow (with the active site nucleophile)-alpha-beta structure, residues 131-175 of the synthase. A conserved histidine of the Class III PHB synthases aligns with the active site histidine of the lipases using the ClustalW algorithm. This is intriguing as this histidine is approximately 200 amino acids removed in sequence space from the catalytic nucleophile. Different threading algorithms suggest that the Class III synthases belong to the alpha/beta hydrolase superfamily which includes prokaryotic lipases. Mutagenesis studies were carried out on C. vinosum synthase C149, H331, H303, D302, and C130 residues. These studies reveal that H331 is the general base catalyst that activates the nucleophile, C149, for covalent catalysis. The model indicates that C130 is not involved in catalysis as previously proposed [Müh, U., Sinskey, A. J., Kirby, D. P., Lane, W. S., and Stubbe, J. (1999) Biochemistry 38, 826-837]. Studies with D302 mutants suggest D302 functions as a general base catalyst in activation of the 3-hydroxyl of HBCoA (or a hydroxybutyrate acyl enzyme) for nucleophilic attack on the covalently linked thiol ester intermediate. The relationship of the lipase model to previous models based on fatty acid synthases is discussed.     

Class I and III polyhydroxyalkanoate synthases from Ralstonia eutropha and Allochromatium vinosum: characterization and substrate specificity studies

Class I and III polyhydroxyalkanoate (PHA) synthases catalyze the conversion of beta-hydroxybutyryl coenzyme A (HBCoA) to polyhydroxybutyrate. The Class I PHA synthase from Ralstonia eutropha has been purified by numerous labs with reported specific activities that vary between 1 and 160 U/mg. An N-terminal (His)6-PHA synthase was constructed and purified with specific activity of 40 U/mg. The variable activity is shown to be related to the protein's propensity to aggregate and not to incomplete post-translational modification by coenzyme A and a phosphopantetheinyl transferase. The substrate specificities of this enzyme and the Class III PHA synthase from Allochromatium vinosum have been determined with nine analogs of varied chain length and branching, OH group position within the chain, and thioesters. The results suggest that in vitro, both PHA synthases are very specific and provide further support for their active site structural similarities. In vitro results differ from studies in vivo.     

Detection of intermediates from the polymerization reaction catalyzed by a D302A mutant of class III polyhydroxyalkanoate (PHA) synthase

Polyhydroxybutyrate (PHB) synthases catalyze the polymerization of (R)-3-hydroxybutyryl-CoA (HB-CoA) into high molecular weight PHB, biodegradable polymers. The class III PHB synthase from Allochromatium vinosum is composed of a 1:1 mixture of two approximately 40 kDa proteins: PhaC and PhaE. Previous studies using site-directed mutagenesis and a saturated trimer of hydroxybutyryl-CoA have suggested the importance of C149 (in covalent catalysis), H331 (in activation of C149), and D302 (in hydroxyl group activation for ester bond formation) in the polymerization process. All three residues are located on PhaC. We now report that incubation of D302A-PhaCPhaE with [14C]-HB-CoA results in detection, for the first time, of oligomeric HBs covalently bound to PhaC. The reaction products have been analyzed by SDS-PAGE, Westerns with PhaCPhaE antibodies, and autoradiography. Different migratory properties of D302A-PhaC on SDS-PAGE have been observed at [14C]-HB-CoA to enzyme (S/E) ratios between 5 and 100. Trypsin digestion and HPLC analysis of the D302A-PhaCPhaE (from a reaction with a S/E ratio of 5) allowed isolation of multiple radiolabeled peptides. N-Terminal sequencing, MALDI-TOF, and ESI mass spectrometric analysis of these peptides revealed that all of the peptides were identical but were modified by (HB)n ranging in size from n = 3 to n = 10. The in vitro results support the role of D302 in elongation rather than in activating the active site cysteine for acylation. This proposal has been further supported by our in vivo studies on a Wautersia eutropha strain in which the class I synthase gene has been replaced with the D302A-PhaCPhaE gene and the organism examined under PHB production conditions by transmission electron microscopy. Very small granules (<0.05 microm) were observed in contrast to the 0.2-0.5 microm granules observed with the wt strain. Use of the D302A synthase has allowed successful interrogation of the initiation and elongation steps catalyzed by the class III synthase.     

Class III polyhydroxybutyrate synthase: involvement in chain termination and reinitiation

Polyhydroxybutyrate (PHB) synthase catalyzes the polymerization of (R)-3-hydroxybutyryl-CoA (CoA = coenzyme A) into high molecular weight PHB. Recombinant wild-type (wt) class III synthase from Allochromatium vinosum (PhaCPhaE(Av)), antibodies to this synthase and to PHB, and [(14)C]hydroxybutyryl-CoA (HB-CoA) have been used to detect oligomeric hydroxybutyrate (HB) units covalently bound to the synthase using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Although a distribution of products is typically observed, short (HB)(n)-bound synthases (designated species I) are most prevalent at low substrate to enzyme (S/E) ratios. Species I is similar to (HB)(n)-PhaC(Av) (n = 3-10 at minimum) recently identified using D302A-PhaCPhaE(Av) (Tian, J., Sinskey, A. J., and Stubbe, J. (2005) Biochemistry 44, 1495-1503). Species I is shown to be an intermediate in the elongation process of PHB synthesis in vitro. The reaction catalyzed by the wt synthase in vitro was further studied under two sets of conditions: at high (70000) and low (<200) S/E ratios. At high S/E ratios, kinetic analysis of the reaction of HB-CoA with the wt synthase monitored using antibodies to PhaCPhaE(Av) and Western blotting revealed the disappearance of PhaC(Av) at early time points and its reappearance as the molecular weight of the PHB approached 1.8 MDa. At low S/E ratios, species I was observed to increase with time after complete consumption of all of the HB-CoA. The results from studies under both sets of conditions suggest that an inherent property of the synthase is chain termination and reinitiation.     

Mechanism of the polymerization reaction initiated and catalyzed by the polyhydroxybutyrate synthase of Ralstonia eutropha

Polyhydroxybutyrate (PHB) synthases (polymerases) catalyze the polymerization of the coenzyme A thioester of 3-hydroxybutyrate to PHB. The Ralstonia eutropha PHB synthase purified from recombinant E. coli cells exists in aqueous solution in both monomeric (single subunit) and homodimeric (two subunits) forms in equilibrium. Several lines of evidence suggest that the homodimer is the active form of the synthase. The initial mechanistic model for the polymerization reaction proposed that two different thiol groups form the catalytic site. The cysteine at 319 has been shown to provide one thiol group that is involved in the covalent catalysis, but a second thiol group on the same protein molecule has not yet been identified. It is suggested that cysteines at 319 from each of the two molecules of a homodimer synthase provide two identical thiol groups to jointly form a single catalytic site. To verify this model using the strategy of in vitro reconstitution, heterodimers composed of the wild-type subunit and of the C(319) mutated subunit were constructed and the activities at various ratios of the wild-type subunit to the mutated subunit were measured. The experimental results indicate that the homodimer is the active form of the enzyme, that the heterodimer containing the mutated subunit has no activity, and that a single cysteine is not sufficient for catalysis. Two identical thiol groups from C(319) residues on each subunit of the homodimer are required to form the catalytic site for the initiation and propagation reactions. We further demonstrate that a dimer synthase that has initiated the polymerization reaction (primed synthase) is significantly more stable against dissociation than the unprimed (unreacted) dimer synthase. These two properties explain the nature of lag phenomenon during the in vitro polymerization reaction catalyzed by this enzyme     

Probing the role of metal ions in the catalysis of Helicobacter pylori 3-deoxy-D-manno-octulosonate-8-phosphate synthase using a transient kinetic analysis

3-Deoxy-d-manno-2-octulosonate-8-phosphate (KDO8P) synthase catalyzes the net condensation of phosphoenolpyruvate and d-arabinose 5-phosphate to form KDO8P and inorganic phosphate (Pi). Two classes of KDO8P synthases have been identified. The Class I KDO8P synthases (e.g. Escherchia coli KDO8P synthase) catalyze the condensation reaction in a metal-independent fashion, whereas the Class II enzymes (e.g. Aquifex aeolicus) require metal ions for catalysis. Helicobacter pylori (H. pylori) KDO8P synthase, a Zn2+-dependent metalloenzyme, has recently been found to be a Class II enzyme and has a high degree of clinical significance since it is an attractive molecular target for the design of novel antibiotic therapy. Although the presence of a divalent metal ion in Class II KDO8P synthases is essential for catalysis, there is a paucity of mechanistic information on the role of the metal ions and functional differences as compared with Class I enzymes. Using H. pylori KDO8P synthase as a prototypical Class II enzyme, a steady-state and transient kinetic approach was undertaken to understand the role of the metal ion in catalysis and define the kinetic reaction pathway. Metal reconstitution experiments examining the reaction kinetics using Zn2+, Cd2+, Cu2+, Co2+, Mn2+, and Ni2+ yielded surprising results in that the Cd2+ enzyme has the greatest activity. Unlike Class-I KDO8P synthases, the Class II metallo-KDO8P synthases containing Zn2+, Cd2+, Cu2+, and Co2+ show cooperativity. This study presents the first detailed kinetic characterization of a metal-dependent Class II KDO8P synthase and offers mechanistic insight for how the divalent metal ions modulate catalysis through effects on chemistry as well as quaternary protein structure.     

Poly(hydroxyalkanoic acid) Biosynthesis in Ectothiorhodospirashaposhnikovii: Characterization and Reactivity of a Type III PHA Synthase

Ectothiorhodospira shaposhnikovii is able to accumulate polyhydroxybutyrate (PHB) photoautotrophically during nitrogen-limited growth. The activity of polyhydroxyalkanoate (PHA) synthase in the cells correlates with PHB accumulation. PHA synthase samples collected during the light period do not show a lag phase during in vitro polymerization. Synthase samples collected in the dark period displays a significant lag phase during in vitro polymerization. The lag phase can be eliminated by reacting the PHA synthase with the monomer, 3-hydroxybutyryl-CoA (3HBCoA). The PHA synthase genes (phaC and phaE) were cloned by screening a genomic library for PHA accumulation in E. coli cells. The PHA synthase expressed in the recombinant E. coli cells was purified to homogeneity. Both sequence analysis and biochemical studies indicated that this PHA synthase consists of two subunits, PhaE and PhaC and, therefore, belongs to the type III PHA synthases. Two major complexes were identified in preparations of purified PHA synthase. The large complex appears to be composed of 12 PhaC subunits and 12 PhaE subunits (dodecamer), whereas the small complex appears to be composed of 6 PhaC and 6 PhaE subunits (hexamer). In dilute aqueous solution, the synthase is predominantly composed of hexamer and has low activity accompanied with a significant lag period at the initial stage of reaction. The percentage of dodecameric complex increases with increasing salt concentration. The dodecameric complex has a greatly increased specific activity for the polymerization of 3HBCoA and a negligible lag period. The results from in vitro polymerizations of 3HBCoA suggest that the PHA synthase from E. shaposhnikovii may catalyze a living polymerization and demonstrate that two PhaC and two PhaE subunits comprise a single catalytic site in the synthase complex.     

Polyhydroxyalkanoate (PHA) accumulation in sulfate-reducing bacteria and identification of a class III PHA synthase (PhaEC) in Desulfococcus multivorans

Seven strains of sulfate-reducing bacteria (SRB) were tested for the accumulation of polyhydroxyalkanoates (PHAs). During growth with benzoate Desulfonema magnum accumulated large amounts of poly(3-hydroxybutyrate) [poly(3HB)]. Desulfosarcina variabilis (during growth with benzoate), Desulfobotulus sapovorans (during growth with caproate), and Desulfobacterium autotrophicum (during growth with caproate) accumulated poly(3HB) that accounted for 20 to 43% of cell dry matter. Desulfobotulus sapovorans and Desulfobacterium autotrophicum also synthesized copolyesters consisting of 3-hydroxybutyrate and 3-hydroxyvalerate when valerate was used as the growth substrate. Desulfovibrio vulgaris and Desulfotalea psychrophila were the only SRB tested in which PHAs were not detected. When total DNA isolated from Desulfococcus multivorans and specific primers deduced from highly conserved regions of known PHA synthases (PhaC) were used, a PCR product homologous to the central region of class III PHA synthases was obtained. The complete pha locus of Desulfococcus multivorans was subsequently obtained by inverse PCR, and it contained adjacent phaE(Dm) and phaC(Dm) genes. PhaC(Dm) and PhaE(Dm) were composed of 371 and 306 amino acid residues and showed up to 49 or 23% amino acid identity to the corresponding subunits of other class III PHA synthases. Constructs of phaC(Dm) alone (pBBRMCS-2::phaC(Dm)) and of phaE(Dm)C(Dm) (pBBRMCS-2::phaE(Dm)C(Dm)) in various vectors were obtained and transferred to several strains of Escherichia coli, as well as to the PHA-negative mutants PHB(-)4 and GPp104 of Ralstonia eutropha and Pseudomonas putida, respectively. In cells of the recombinant strains harboring phaE(Dm)C(Dm) small but significant amounts (up to 1.7% of cell dry matter) of poly(3HB) and of PHA synthase activity (up to 1.5 U/mg protein) were detected. This indicated that the cloned genes encode functionally active proteins. Hybrid synthases consisting of PhaC(Dm) and PhaE of Thiococcus pfennigii or Synechocystis sp. strain PCC 6308 were also constructed and were shown to be functionally active.     

Functional importance of motif I of pseudouridine synthases: mutagenesis of aligned lysine and proline residues

On the basis of sequence alignments, the pseudouridine synthases were grouped into four families that share no statistically significant global sequence similarity, though some common sequence motifs were discovered [Koonin, E. V. (1996) Nucleic Acids. Res. 24, 2411-2415; Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762]. We have investigated the functional significance of these alignments by substituting the nearly invariant lysine and proline residues in Motif I of RluA and TruB, pseudouridine synthases belonging to different families. Contrary to our expectations, the altered enzymes display only very mild kinetic impairment. Substitution of the aligned lysine and proline residues does, however, reduce structural stability, consistent with a temperature sensitive phenotype that results from substitution of the cognate proline residue in Cbf5p, a yeast homologue of TruB [Zerbarjadian, Y., King, T., Fournier, M. J., Clarke, L., and Carbon, J. (1999) Mol. Cell. Biol. 19, 7461-7472]. Together, our data support a functional role for Motif I, as predicted by sequence alignments, though the effect of substituting the highly conserved residues was milder than we anticipated. By extrapolation, our findings also support the assignment of pseudouridine synthase function to certain physiologically important eukaryotic proteins that contain Motif I, including the human protein dyskerin, alteration of which leads to the disease dyskeratosis congenita.     

Purification of polyhydroxybutyrate synthase from its native organism, Ralstonia eutropha: implications for the initiation and elongation of polymer formation in vivo

Class I polyhydroxybutyrate (PHB) synthase (PhaC) from Ralstonia eutropha catalyzes the formation of PHB from (R)-3-hydroxybutyryl-CoA, ultimately resulting in the formation of insoluble granules. Previous mechanistic studies of R. eutropha PhaC, purified from Escherichia coli (PhaC(Ec)), demonstrated that the polymer elongation rate is much faster than the initiation rate. In an effort to identify a factor(s) from the native organism that might prime the synthase and increase the rate of polymer initiation, an N-terminally Strep2-tagged phaC (Strep2-PhaC(Re)) was constructed and integrated into the R. eutropha genome in place of wild-type phaC. Strep2-PhaC(Re) was expressed and purified by affinity chromatography from R. eutropha grown in nutrient-rich TSB medium for 4 h (peak production PHB, 15% cell dry weight) and 24 h (PHB, 2% cell dry weight). Analysis of the purified PhaC by size exclusion chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gel permeation chromatography revealed that it unexpectedly copurified with the phasin protein, PhaP1, and with soluble PHB (M(w) = 350 kDa) in a "high-molecular weight" (HMW) complex and in monomeric/dimeric (M/D) forms with no associated PhaP1 or PHB. Assays for monitoring the formation of PHB in the HMW complex showed no lag phase in CoA release, in contrast to M/D forms of PhaC(Re) (and PhaC(Ec)), suggesting that PhaC in the HMW fraction has been isolated in a PHB-primed form. The presence of primed and nonprimed PhaC suggests that the elongation rate for PHB formation is also faster than the initiation rate in vivo. A modified micelle model for granule genesis is proposed to accommodate the reported observations.     

Polyhydroxyalkanoate (PHA) synthesis by class IV PHA synthases employing Ralstonia eutropha PHB(-)4 as host strain

Class IV polyhydroxyalkanoate (PHA) synthase from Bacillus cereus YB-4 (PhaRC(YB4)) or B. megaterium NBRC15308(T) (PhaRC(Bm)) was expressed in Ralstonia eutropha PHB(-)4 to compare the ability to produce PHA and the substrate specificity of PhaRCs. PhaRC(YB4) produced significant amounts of PHA and had broader substrate specificity than PhaRC(Bm).     

Lysine221 is the general base residue of the isochorismate synthase from Pseudomonas aeruginosa (PchA) in a reaction that is diffusion limited

The isochorismate synthase from Pseudomonas aeruginosa (PchA) catalyzes the conversion of chorismate to isochorismate, which is subsequently converted by a second enzyme (PchB) to salicylate for incorporation into the salicylate-capped siderophore pyochelin. PchA is a member of the MST family of enzymes, which includes the structurally homologous isochorismate synthases from Escherichia coli (EntC and MenF) and salicylate synthases from Yersinia enterocolitica (Irp9) and Mycobacterium tuberculosis (MbtI). The latter enzymes generate isochorismate as an intermediate before generating salicylate and pyruvate. General acid–general base catalysis has been proposed for isochorismate synthesis in all five enzymes, but the residues required for the isomerization are a matter of debate, with both lysine221 and glutamate313 proposed as the general base (PchA numbering). This work includes a classical characterization of PchA with steady state kinetic analysis, solvent kinetic isotope effect analysis and by measuring the effect of viscosogens on catalysis. The results suggest that isochorismate production from chorismate by the MST enzymes is the result of general acid–general base catalysis with a lysine as the base and a glutamic acid as the acid, in reverse protonation states. Chemistry is determined to not be rate limiting, favoring the hypothesis of a conformational or binding step as the slow step.     

Comparative analysis of folding and substrate binding sites between regulated hexameric type II citrate synthases and unregulated dimeric type I enzymes.

We describe the first structure determination of a type II citrate synthase, an enzyme uniquely found in Gram-negative bacteria. Such enzymes are hexameric and are strongly and specifically inhibited by NADH through an allosteric mechanism. This is in contrast to the widespread dimeric type I citrate synthases found in other organisms, which do not show allosteric properties. Our structure of the hexameric type II citrate synthase from Escherichia coli is composed of three identical dimer units arranged about a central 3-fold axis. The interactions that lead to hexamer formation are concentrated in a relatively small region composed of helix F, FG and IJ helical turns, and a seven-residue loop between helices J and K. This latter loop is present only in type II citrate synthase sequences. Running through the middle of the hexamer complex, and along the 3-fold axis relating dimer units, is a remarkable pore lined with 18 cationic residues and an associated hydrogen-bonded network. Also unexpected was the observation of a novel N-terminal domain, formed by the collective interactions of the first 52 residues from the two subunits of each dimer. The domain formed is rich in beta-sheet structure and has no counterpart in previous structural studies of type I citrate synthases. This domain is located well away from the dimer-dimer contacts that form the hexamer, and it is not involved in hexamer formation. Another surprising observation from the structure of type II E. coli citrate synthase is the unusual polypeptide chain folding found at the putative acetylcoenzyme A binding site. Key parts of this region, including His264 and a portion of polypeptide chain known from type I structures to form an adenine binding loop (residues 299-303), are shifted by as much as 10 A from where they must be for substrate binding and catalysis to occur. Furthermore, the adjacent polypeptide chain composed of residues 267-297 is extremely mobile in our structure. Thus, acetylcoenzyme A binding to type II E. coli citrate synthase would require substantial structural shifts and a concerted refolding of the polypeptide chain to form an appropriate binding subsite. We propose that this essential rearrangement of the acetylcoenzyme A binding part of the active site is also a major feature of allostery in type II citrate synthases. Overall, this study suggests that the evolutionary development of hexameric association, the elaboration of a novel N-terminal domain, introduction of a NADH binding site, and the need to refold a key substrate binding site are all elements that have been developed to allow for the allosteric control of catalysis in the type II citrate synthases.     

Morphological changes induced by class III chitin synthase gene silencing could enhance penicillin production of Penicillium chrysogenum

Chitin synthases catalyze the formation of β-(1,4)-glycosidic bonds between N-acetylglucosamine residues to form the unbranched polysaccharide chitin, which is the major component of cell walls in most filamentous fungi. Several studies have shown that chitin synthases are structurally and functionally divergent and play crucial roles in the growth and morphogenesis of the genus Aspergillus although little research on this topic has been done in Penicillium chrysogenum. We used BLAST to find the genes encoding chitin synthases in P. chrysogenum related to chitin synthase genes in Aspergillus nidulans. Three homologous sequences coding for a class III chitin synthase CHS4 and two hypothetical proteins in P. chrysogenum were found. The gene which product showed the highest identity and encoded the class III chitin synthase CHS4 was studied in detail. To investigate the role of CHS4 in P. chrysogenum morphogenesis, we developed an RNA interference system to silence the class III chitin synthase gene chs4. After transformation, mutants exhibited a slow growth rate and shorter and more branched hyphae, which were distinct from those of the original strain. The results also showed that the conidiation efficiency of all transformants was reduced sharply and indicated that chs4 is essential in conidia development. The morphologies of all transformants and the original strain in penicillin production were investigated by light microscopy, which showed that changes in chs4 expression led to a completely different morphology during fermentation and eventually caused distinct penicillin yields, especially in the transformants PcRNAi1-17 and PcRNAi2-1 where penicillin production rose by 27 % and 41 %, respectively.     

Implications of secondary structure prediction and amino acid sequence comparison of class I and class II phosphoribosyl diphosphate synthases on catalysis, regulation, and quaternary structure

Spinach 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) synthase isozyme 4 was synthesized in Escherichia coli and purified to near homogeneity. The activity of the enzyme is independent of P(i); it is inhibited by ADP in a competitive manner, indicating a lack of an allosteric site; and it accepts ATP, dATP, GTP, CTP, and UTP as diphosphoryl donors. All of these properties are characteristic for class II PRPP synthases. K(m) values for ATP and ribose 5-phosphate are 77 and 48 microM, respectively. Gel filtration reveals a molecular mass of the native enzyme of approximately 110 kD, which is consistent with a homotrimer. Secondary structure prediction shows that spinach PRPP synthase isozyme 4 has a general folding similar to that of Bacillus subtilis class I PRPP synthase, for which the three-dimensional structure has been solved, as the position and extent of helices and beta-sheets of the two enzymes are essentially conserved. Amino acid sequence comparison reveals that residues of class I PRPP synthases interacting with allosteric inhibitors are not conserved in class II PRPP synthases. Similarly, residues important for oligomerization of the B. subtilis enzyme show little conservation in the spinach enzyme. In contrast, residues of the active site of B. subtilis PRPP synthase show extensive conservation in spinach PRPP synthase isozyme 4.     

Thiolysis of poly(3-hydroxybutyrate) with polyhydroxyalkanoate synthase from Ralstonia eutropha

Poly(3-hydroxybutyrate) (PHB) is synthesized from 3-hydroxybutyryl-CoA by polyhydroxyalkanoate synthase and hydrolyzed by PHB depolymerase. In this study, we focused on the reverse reaction of polyhydroxyalkanoate synthase, and propose the possibility that PHB can be degraded through a novel process, that is thiolysis of PHB with CoASH. Polyhydroxyalkanoate synthase of Ralstonia eutropha was incubated with 14C-labeled PHB and CoASH. The reaction mixture was fractionated by HPLC and then analyzed with a scintillation counter. The analysis revealed 3-hydroxybutyryl-CoA to be a product of the reaction. When NADP+ and acetoacetyl-CoA reductase were added to the reaction mixture, an increase in absorbance at 340 nm was observed. Native PHB inclusion bodies from R. eutropha also showed thiolytic activity. This is the first indication that polyhydroxyalkanoate synthase catalyzes both the synthesis and degradation of PHB, and that native PHB inclusion bodies has thiolytic activity.     

Mutation on N-terminus of polyhydroxybutyrate synthase of Ralstonia eutropha enhanced PHB accumulation

Polyhydroxyalkanoate (PHA) synthase is the central enzyme involved in the biosynthesis of PHA, a family of bacterial biodegradable polyesters. Due to its high variability, the N-terminal fragment of this enzyme was previously considered as unnecessary for a functionally active enzyme. In this study, polyhydroxybutyrate synthase from Ralstonia eutropha (PhbC(Re)) with a deletion on N-terminal 88 amino acid residues showed a significant reduced activity, as reflected by only 1.5% PHB accumulation compared with the wild type which produced 58.4% PHB of the cell dry weight. Whilst several site-specific mutagenesis results revealed the amphiphilic alpha-helix assembled by the amino acid region, D70-E88 played an important role in both maintaining the PHB synthase activity and regulating molecular weight and polydispersity of accumulated PHB homopolymer.     

Amino acid binding by the class I aminoacyl-tRNA synthetases: role for a conserved proline in the signature sequence.

Although partial or complete three-dimensional structures are known for three Class I aminoacyl-tRNA synthetases, the amino acid-binding sites in these proteins remain poorly characterized. To explore the methionine binding site of Escherichia coli methionyl-tRNA synthetase, we chose to study a specific, randomly generated methionine auxotroph that contains a mutant methionyl-tRNA synthetase whose defect is manifested in an elevated Km for methionine (Barker, D.G., Ebel, J.-P., Jakes, R.C., & Bruton, C.J., 1982, Eur. J. Biochem. 127, 449-457), and employed the polymerase chain reaction to sequence this mutant synthetase directly. We identified a Pro 14 to Ser replacement (P14S), which accounts for a greater than 300-fold elevation in Km for methionine and has little effect on either the Km for ATP or the kcat of the amino acid activation reaction. This mutation destabilizes the protein in vivo, which may partly account for the observed auxotrophy. The altered proline is found in the "signature sequence" of the Class I synthetases and is conserved. This sequence motif is 1 of 2 found in the 10 Class I aminoacyl-tRNA synthetases and, in the known structures, it is in the nucleotide-binding fold as part of a loop between the end of a beta-strand and the start of an alpha-helix. The phenotype of the mutant and the stability and affinity for methionine of the wild-type and mutant enzymes are influenced by the amino acid that is 25 residues beyond the C-terminus of the signature sequence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular characterization of the poly(3-hydroxybutyrate) (PHB) synthase from Ralstonia eutropha: in vitro evolution, site-specific mutagenesis and development of a PHB synthase protein model

A threading model of the Ralstonia eutropha polyhydroxyalkanoate (PHA) synthase was developed based on the homology to the Burkholderia glumae lipase, whose structure has been resolved by X-ray analysis. The lid-like structure in the model was discussed. In this study, various R. eutropha PHA synthase mutants were generated employing random as well as site-specific mutagenesis. Four permissive mutants (double and triple mutations) were obtained from single gene shuffling, which showed reduced activity and whose mutation sites mapped at variable surface-exposed positions. Six site-specific mutations were generated in order to identify amino acid residues which might be involved in substrate specificity. Replacement of residues T323 (I/S) and C438 (G), respectively, which are located in the core structure of the PHA synthase model, abolished PHA synthase activity. Replacement of the two amino acid residues Y445 (F) and L446 (K), respectively, which are located at the surface of the protein model and adjacent to W425, resulted in reduced activity without changing substrate specificity and indicating a functional role of these residues. The E267K mutant exhibited only slightly reduced activity with a surface-exposed mutation site. Four site-specific deletions were generated to evaluate the role of the C-terminus and variant amino acid sequence regions, which link highly conserved regions. Deleted regions were D281-D290, A372-C382, E578-A589 and V585-A589 and the respective PHA synthases showed no detectable activity, indicating an essential role of the variable C-terminus and the linking regions between conserved blocks 2 and 3 as well as 3 and 4. Moreover, the N-terminal part of the class II PHA synthase (PhaC(Pa)) from Pseudomonas aeruginosa and the C-terminal part of the class I PHA synthase (PhaC(Re)) from R. eutropha were fused, respectively, resulting in three fusion proteins with no detectable in vivo activity. However, the fusion protein F1 (PhaC(Pa)-1-265-PhaC(Re)-289-589) showed 13% of wild type in vitro activity with the fusion point located at a surface-exposed loop region.     

Poly(3-hydroxybutyrate) synthesis genes in Azotobacter sp. strain FA8.

Genes responsible for the synthesis of poly(3-hydroxybutyrate) (PHB) in Azotobacter sp. FA8 were cloned and analyzed. A PHB polymerase gene (phbC) was found downstream from genes coding for beta-ketothiolase (phbA) and acetoacetyl-coenzyme A reductase (phbB). A PHB synthase mutant was obtained by gene inactivation and used for genetic studies. The phbC gene from this strain was introduced into Ralstonia eutropha PHB-4 (phbC-negative mutant), and the recombinant accumulated PHB when either glucose or octanoate was used as a source of carbon, indicating that this PHB synthase cannot incorporate medium-chain-length hydroxyalkanoates into PHB.