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.     

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.     

Isolated poly(3-hydroxybutyrate) (PHB) granules are complex bacterial organelles catalyzing formation of PHB from acetyl coenzyme A (CoA) and degradation of PHB to acetyl-CoA

Poly(3-hydroxybutyrate) (PHB) granules isolated in native form (nPHB granules) from Ralstonia eutropha catalyzed formation of PHB from ¹⁴C-labeled acetyl coenzyme A (CoA) in the presence of NADPH and concomitantly released CoA, revealing that PHB biosynthetic proteins (acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase, and PHB synthase) are present and active in isolated nPHB granules in vitro. nPHB granules also catalyzed thiolytic cleavage of PHB in the presence of added CoA, resulting in synthesis of 3-hydroxybutyryl-CoA (3HB-CoA) from PHB. Synthesis of 3HB-CoA was also shown by incubation of artificial (protein-free) PHB with CoA and PhaZa1, confirming that PhaZa1 is a PHB depolymerase catalyzing the thiolysis reaction. Acetyl-CoA was the major product detectable after incubation of nPHB granules in the presence of NAD⁺, indicating that downstream mobilizing enzyme activities were also present and active in isolated nPHB granules. We propose that intracellular concentrations of key metabolites (CoA, acetyl-CoA, 3HB-CoA, NAD⁺/NADH) determine whether a cell accumulates or degrades PHB. Since the degradation product of PHB is 3HB-CoA, the cells do not waste energy by synthesis and degradation of PHB. Thus, our results explain the frequent finding of simultaneous synthesis and breakdown of PHB.     

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     

In vitro evolution of a polyhydroxybutyrate synthase by intragenic suppression-type mutagenesis

In vitro evolution was applied to obtain highly active mutants of Ralstonia eutropha polyester synthase (PhbC(Re)), which is a key enzyme catalyzing the formation of polyhydroxybutyrate (PHB) from (R)-3-hydroxybutyryl-CoA (3HB-CoA). To search for beneficial mutations for activity improvement of this enzyme, we have conducted multi-step mutations, including activity loss and intragenic suppression-type activity reversion. Among 259 revertants, triple mutant E11S12 was obtained as the most active one via PCR-mediated secondary mutagenesis from mutant E11 with a single mutation (Ser to Pro at position 80), which exhibited reduced activity (as low as 27% of the wild-type level) but higher thermostability compared to the wild-type enzyme. Mutant E11S12 exhibited up to 79% of the wild-type enzyme activity. Mutation separation of E11S12 revealed that the replacement of Phe by Ser at position 420 (F420S), located in a highly conserved alpha/beta hydrolase fold region, of the E11S12 mutant contributes to the improvement of the enzyme activity. A purified sample of the genetically engineered mutant, termed E11S12-1, with the F420S mutation alone was found to exhibit a 2.4-fold increase in specific activity toward 3HB-CoA, compared to the wild-type.     

Poly(3-Hydroxybutyrate) Degradation in Ralstonia eutropha H16 Is Mediated Stereoselectively to (S)-3-Hydroxybutyryl Coenzyme A (CoA) via Crotonyl-CoA

Degradation of poly(3-hydroxybutyrate) (PHB) by the thiolytic activity of the PHB depolymerase PhaZ1 from Ralstonia eutropha H16 was analyzed in the presence of different phasins. An Escherichia coli strain was constructed that harbored the genes for PHB synthesis (phaCAB), the phasin PhaP1, and the PHB depolymerase PhaZ1. PHB was isolated in the native form (nPHB) from this recombinant E. coli strain, and the in vitro degradation of the polyester was examined. Degradation resulted in the formation of the expected 3-hydroxybutyryl coenzyme A (3HB-CoA) and in the formation of a second product, which occurred in significantly higher concentrations than 3HB-CoA. This second product was identified by liquid chromatography mass spectrometry (LC-MS) as crotonyl-CoA. Replacement of PhaP1 by PhaP2 or PhaP4 resulted in a lower degradation rate, whereas the absence of the phasins prevented the degradation of nPHB by the PHB depolymerase PhaZ1 almost completely. In addition, the in vitro degradation of nPHB granules isolated from R. eutropha H16 (wild type) and from the R. eutropha ΔphaP1 and ΔphaP1-4 deletion mutants was examined. In contrast to the results obtained with nPHB granules isolated from E. coli, degradation of nPHB granules isolated from the wild type of R. eutropha yielded high concentrations of 3HB-CoA and low concentrations of crotonyl-CoA. The degradation of nPHB granules isolated from the ΔphaP1 and ΔphaP1-4 deletion mutants of R. eutropha was significantly reduced in comparison to that of nPHB granules isolated from wild-type R. eutropha. Stereochemical analyses of 3HB-CoA revealed that the (R) stereoisomer was collected after degradation of granules isolated from E. coli, whereas the (S) stereoisomer was collected after degradation of granules isolated from R. eutropha. Based on these results, a newly observed mechanism in the degradation pathway for PHB in R. eutropha is proposed which is connected by crotonyl-CoA to the β-oxidation cycle. According to this model, the NADPH-dependent synthesis of PHB with (R)-3HB-CoA as the intermediate and the PHB degradation yielding (S)-3HB-CoA, which is further converted in an NAD-dependent reaction, are separated.     

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.     

Detection of polyhydroxyalkanoate synthase activity on a polyacrylamide gel

This study presents a method to detect active polyhydroxyalkanoate (PHA) synthase on a polyacrylamide gel that combines the polyhydroxybutyrate (PHB) polymerization reaction with Sudan Black B staining. After separation of the protein samples on a modified sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the slab gel was submerged in a buffer containing β-hydroxybutyryl-coenzyme A (3-HBCoA) as substrate and incubated at room temperature for in vitro PHB polymerization. The active PHA synthase catalyzed 3-HBCoA into the PHB polymer and was stained with Sudan Black B. The active PHA synthase appeared as a dark blue band. The activity staining was of high sensitivity, capable of detecting 3.9 ng (0.273 mU) of Cupriavidus necator H16 PHA synthase purified from recombinant Escherichia coli. The detection sensitivity of activity staining was comparable to that of Western blotting analysis. Furthermore, the high sensitivity of activity staining enabled specific detection of the active PHA synthase in the crude extract of wild-type strain C. necator H16. This study provides a rapid, sensitive, and highly specific method for detecting active PHA synthase in gel. The method could be applied to detecting PHA synthase from wild-type bacteria and to the process of enzyme purification.     

Atomic force microscopic observation of in vitro polymerized poly[(R)-3-hydroxybutyrate]: insight into possible mechanism of granule formation

Atomic force microscopy (AFM) was used to study the formation and growth of poly[(R)-3-hydroxybutyrate] (PHB) structures formed in the enzymatic polymerization of (R)-3-hydroxybutyryl coenzyme A [(R)-3-HBCoA] in vitro. Poly(3-hydroxyalkanoate) (PHA) synthase (PhaC(Re)) from Ralstonia eutropha, a class I synthase, was purified by one-step purification and then used for in vitro reactions. Before the reaction, PhaC(Re) molecules were deposited on highly oriented pyrolytic graphite (HOPG) and observed as spherical particles with an average height of 2.7 +/- 0.6 nm and apparent width of 24 +/- 3 nm. AFM analysis during the initial stage of the reaction, that is, after a small amount of (R)-3-HBCoA had been consumed, showed that the enzyme molecules polymerize (R)-3-HBCoA and form flexible 3HB polymer chains that extend from the enzyme particles, resulting in the formation of an enzyme-nascent PHB conjugate. When a sufficient amount of (R)-3-HBCoA was used as substrate, the reaction rapidly increased after the first minute followed by a slow increase in rate, and substrate was completely consumed after 4 min. After 4 min, spherical granules continued to grow in size to form clusters over 10 um in width, and in later stages of cluster formation, the cluster developed small projections with a size of approximately 100-250 nm, suggesting qualitative changes of the PHB clusters. Moreover, the high-resolution AFM images suggested that globular structures of approximately 20-30 nm apparent width, which corresponds to the size of PhaC(Re), were located on the surface of the small PHB granule particles.     

Alteration of substrate chain-length specificity of type II synthase for polyhydroxyalkanoate biosynthesis by in vitro evolution: in vivo and in vitro enzyme assays

In our previous study, in vitro evolution of type II polyhydroxyalkanoate (PHA) synthase (PhaC1Ps) from Pseudomonas sp. 61-3 yielded eleven mutant enzymes capable of synthesizing homopolymer of (R)-3-hydroxybutyrate [P(3HB)] in recombinant Escherichia coli JM109. These recombinant strains were capable of accumulating up to approximately 400-fold more P(3HB) than strains expressing the wild-type enzyme. These mutations enhanced the ability of the enzyme to specifically incorporate the 3HB-coenzyme A (3HB-CoA) substrate or improved catalytic efficiency toward the various monomer substrates of C4 to C12 (R)-3-hydroxyacyl-CoAs which can intrinsically be channeled by PhaC1Ps into P(3HB-co-3HA) copolymerization. In this study, beneficial amino acid substitutions of PhaC1Ps were analyzed based on the accumulation level and the monomer composition of P(3HB-co-3HA) copolymers generated by E. coli LS5218 [fadR601 atoC(Con)] harboring the monomer supplying enzyme genes. Substitutions of Ser by Thr(Cys) at position 325 were found to lead to an increase in the total amount of P(3HB-co-3HA) accumulated, whereas 3HB fractions in the P(3HB-co-3HA) copolymer were enriched by substitutions of Gln by Lys(Arg, Met) at position 481. This strongly suggests that amino acid substitutions at positions 325 and 481 are responsible for synthase activity and/or substrate chain-length specificity of PhaC1Ps. These in vivo results were supported by the in vitro results obtained from synthase activity assays using representative single and double mutants and synthetic substrates, (R,S)-3HB-CoA and (R,S)-3-hydroxydecanoyl-CoA. Notably, the position 481 was found to be a determinant for substrate chain-length specificity of PhaC1Ps.     

PhaM Is the Physiological Activator of Poly(3-Hydroxybutyrate) (PHB) Synthase (PhaC1) in Ralstonia eutropha

Poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) is the key enzyme of PHB synthesis in Ralstonia eutropha and other PHB-accumulating bacteria and catalyzes the polymerization of 3-hydroxybutyryl-CoA to PHB. Activity assays of R. eutropha PHB synthase are characterized by the presence of lag phases and by low specific activity. It is assumed that the lag phase is caused by the time necessary to convert the inactive PhaC1 monomer into the active dimeric form by an unknown priming process. The lag phase can be reduced by addition of nonionic detergents such as hecameg [6-O-(N-heptyl-carbamoyl)-methyl-α-d-glucopyranoside], which apparently accelerates the formation of PhaC1 dimers. We identified the PHB granule-associated protein (PGAP) PhaM as the natural primer (activator) of PHB synthase activity. PhaM was recently discovered as a novel type of PGAP with multiple functions in PHB metabolism. Addition of PhaM to PHB synthase assays resulted in immediate polymerization of 3HB coenzyme A with high specific activity and without a significant lag phase. The effect of PhaM on (i) PhaC1 activity, (ii) oligomerization of PhaC1, (iii) complex formation with PhaC1, and (iv) PHB granule formation in vitro and in vivo was shown by cross-linking experiments of purified proteins (PhaM, PhaC1) with glutardialdehyde, by size exclusion chromatography, and by fluorescence microscopic detection of de novo-synthesized PHB granules.     

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.     

Correlation between structure of the lactones and substrate specificity in enzyme-catalyzed polymerization for the synthesis of polyesters

Small-size (4-membered) and medium-size (5-, 6-, and 7-membered) unsubstituted lactones as well as unsubstituted macrolides (12 and 13 membered) were subjected to the ring-opening polymerization using the extracellular PHB depolymerase from Alcaligenes faecalis T1 (PhaZ(Afa)). The characteristic reactivities of the lactones were discussed based on a tertiary structure model of the active site of the PhaZ(Afa). With respect to the ring-size of the lactones, the 4-membered beta-propiolactone and 6-membered delta-valerolactone (delta-VL) showed the highest polymerization activity, and delta-VL seemed to be the upper size limit for the molecular recognition of the narrow active site cleft of PhaZ(Afa). On the other hand, epsilon-caprolactone, 11-undecanolide, and 12-dodecanolide, which showed excellent polymerization activities by lipases, were scarcely polymerized by PhaZ(Afa). This was ascribed to the difference in the recognition sites between PhaZ(Afa) and lipase. In addition, the effect of the substrate-binding domain of PhaZ(Afa) and the enantioselective ring-opening polymerization of (R,S)-beta-butyrolactone ((R,S)-beta-BL) were studied. The substrate-binding domain lacking PhaZ(Afa) showed higher reactivities than PhaZ(Afa) for the polymerization of the lactones and that a significant enantioselectivity was observed at the early stage of the polymerization of (R,S)-beta-BL to produce the (R)-enriched optically active poly(3-hydroxybutyrate).     

The use of an acetoacetyl‐CoA synthase in place of a β‐ketothiolase enhances poly‐3‐hydroxybutyrate production in sugarcane mesophyll cells

Engineering the production of polyhydroxyalkanoates (PHAs) into high biomass bioenergy crops has the potential to provide a sustainable supply of bioplastics and energy from a single plant feedstock. One of the major challenges in engineering C₄ plants for the production of poly[(R)‐3‐hydroxybutyrate] (PHB) is the significantly lower level of polymer produced in the chloroplasts of mesophyll (M) cells compared to bundle sheath (BS) cells, thereby limiting the full PHB yield‐potential of the plant. In this study, we provide evidence that the access to substrate for PHB synthesis may limit polymer production in M chloroplasts. Production of PHB in M cells of sugarcane is significantly increased by replacing β‐ketothiolase, the first enzyme in the bacterial PHA pathway, with acetoacetyl‐CoA synthase. This novel pathway enabled the production of PHB reaching an average of 6.3% of the dry weight of total leaf biomass, with levels ranging from 3.6 to 11.8% of the dry weight (DW) of individual leaves. These yields are more than twice the level reported in PHB‐producing sugarcane containing the β‐ketothiolase and illustrate the importance of producing polymer in mesophyll plastids to maximize yield. The molecular weight of the polymer produced was greater than 2 × 10⁶ Da. These results are a major step forward in engineering a high biomass C₄ grass for the commercial production of PHB.     

Biosynthesis of polyhydroxyalkanoates (PHA) by recombinant Ralstonia eutropha and effects of PHA synthase activity on in vivo PHA biosynthesis.

Recombinant strains of Ralstonia eutropha PHB 4, which harbored Aeromonas caviae polyhydroxyalkanoates (PHA) biosynthesis genes under the control of a promoter for R. eutropha phb operon, were examined for PHA production from various alkanoic acids. The recombinants produced poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from hexanoate and octanoate, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxypentano ate) [P(3HB-co-3HV-co-3HHp)] from pentanoate and nonanoate. One of the recombinant strains, R. eutropha PHB 4/pJRDBB39d3 harboring ORF1 and PHA synthase gene of A. caviae (phaC(Ac)) accumulated copolyesters with much more 3HHx or 3HHp fraction than the other recombinant strains. To investigate the relationship between PHA synthase activity and in vivo PHA biosynthesis in R. eutropha, the PHB- 4 strains harboring pJRDBB39d13 or pJRDEE32d13 were used, in which the heterologous expression of phaC(Ac) was controlled by promoters for R. eutropha phb operon and A. caviae pha operon, respectively. The PHA contents and PHA accumulation rates were similar between the two recombinant strains in spite of the quite different levels of PHA synthase activity, indicating that the polymerization step is not the rate-determining one in PHA biosynthesis by R. eutropha. The molecular weights of poly(3-hydroxybutyrate) produced by the recombinant strains were also independent of the levels of PHA synthase activity. It has been suggested that a chain-transfer agent is generated in R. eutopha cells to regulate the chain length of polymers.     

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.     

Stereochemistry of catalysis by the ketoreductase activity in the first extension module of the erythromycin polyketide synthase

Multiple ketoreductase activities play a crucial role in establishing the stereochemistry of the products of modular polyketide synthases (PKSs), but there has been little systematic scrutiny of catalysis by individual ketoreductases. To allow this, a diketide synthase, consisting of the loading module, first extension module, and the chain-terminating thioesterase of the erythromycin-producing PKS of Saccharopolyspora erythraea, has been expressed and purified. The DNA encoding the ketoreductase-1 domain in this construct is flanked by unique restriction sites so that another ketoreductase domain can be readily substituted. The purified recombinant diketide synthase catalyzes, at a very low rate (k(cat) equals 2.5 x 10(-3) s(-1)), the specific production of the diketide (2S,3R)-2-methyl-3-hydroxypentanoic acid. The activity of the ketoreductase domain in this model synthase was analyzed using as a model substrate (+/-)-2-methyl-3-oxopentanoic acid N-acetylcysteaminyl (NAC) ester for which k(cat)/K(m) was 21.7 M(-1) s(-1). The NAC thioester of (2S,3R)-2-methyl-3-hydroxypentanoic acid was the major product and was strongly preferred over other stereoisomers as a substrate in the reverse reaction. The bicyclic ketone (9RS)-trans-1-decalone, a known substrate for ketoreductase in fatty acid synthase, was found also to be an effective substrate for the ketoreductase of the diketide synthase. Only the (9R)-trans-1-decalone was reduced, selectively and reversibly, to the (1S,9R)-trans-decalol. The stereochemical course of reduction and oxidation is exactly as found previously for the ketoreductase of animal fatty acid synthase, an additional indication of the close similarity of these enzymes.     

The "intracellular" poly(3-hydroxybutyrate) (PHB) depolymerase of Rhodospirillum rubrum is a periplasm-located protein with specificity for native PHB and with structural similarity to extracellular PHB depolymerases

Rhodospirillum rubrum possesses a putative intracellular poly(3-hydroxybutyrate) (PHB) depolymerase system consisting of a soluble PHB depolymerase, a heat-stable activator, and a 3-hydroxybutyrate dimer hydrolase (J. M. Merrick and M. Doudoroff, J. Bacteriol. 88:60-71, 1964). In this study we reinvestigated the soluble R. rubrum PHB depolymerase (PhaZ1). It turned out that PhaZ1 is a novel type of PHB depolymerase with unique properties. Purified PhaZ1 was specific for amorphous short-chain-length polyhydroxyalkanoates (PHA) such as native PHB, artificial PHB, and oligomer esters of (R)-3-hydroxybutyrate with 3 or more 3-hydroxybutyrate units. Atactic PHB, (S)-3-hydroxybutyrate oligomers, medium-chain-length PHA, and lipase substrates (triolein, tributyrin) were not hydrolyzed. The PHB depolymerase structural gene (phaZ1) was cloned. Its deduced amino acid sequence (37,704 Da) had no significant similarity to those of intracellular PHB depolymerases of Wautersia eutropha or of other PHB-accumulating bacteria. PhaZ1 was found to have strong amino acid homology with type-II catalytic domains of extracellular PHB depolymerases, and Ser(42), Asp(138), and His(178) were identified as catalytic-triad amino acids, with Ser(42) as the putative active site. Surprisingly, the first 23 amino acids of the PHB depolymerase previously assumed to be intracellular revealed features of classical signal peptides, and Edman sequencing of purified PhaZ1 confirmed the functionality of the predicted cleavage site. Extracellular PHB depolymerase activity was absent, and analysis of cell fractions unequivocally showed that PhaZ1 is a periplasm-located enzyme. The previously assumed intracellular activator/depolymerase system is unlikely to have a physiological function in PHB mobilization in vivo. A second gene, encoding the putative true intracellular PHB depolymerase (PhaZ2), was identified in the genome sequence of R. rubrum.     

Effects of recombinant precursor pathway variations on poly[(R)-3-hydroxybutyrate] synthesis in Saccharomyces cerevisiae

Different recombinant R-3-hydroxybutyryl-CoA (3-HB) synthesis pathways strongly influenced the rate and accumulation of the biopolymer poly[(R)-3-hydroxybutyrate] (PHB) in Saccharomyces cerevisiae. It has been previously shown that expression of the Cupriavidus necator PHB synthase gene leads to PHB accumulation in S. cerevisiae [Leaf, T., Peterson, M., Stoup, S., Somers, D., Srienc, F., 1996. Saccharomyces cerevisiae expressing bacterial polyhydroxybutyrate synthase produces poly-3-hydroxybutyrate. Microbiology 142, 1169-1180]. This finding indicates that native S. cerevisiae expresses genes capable of synthesizing the correct stereochemical substrate for the synthase enzyme. The effects of variations of 3-HB precursor pathways on PHB accumulation were investigated by expressing combinations of C. necator PHB pathway genes. When only the PHB synthase gene was expressed, the cells accumulated biopolymer to approximately 0.2% of their cell dry weight. When the PHB synthase and reductase gene were co-expressed, the PHB levels increased approximately 18 fold to about 3.5% of the cell dry weight. When the beta-ketothiolase, reductase and synthase genes were all expressed, the strain accumulated PHB to approximately 9% of the cell dry weight which is 45 fold higher than in the strain with only the synthase gene. Fluorescent microscopic analysis revealed significant cell-to-cell heterogeneity in biopolymer accumulation. While the population average for the strain expressing three PHB genes was approximately 9% of the cell dry weight, some cells accumulated PHB in excess of 50% of their cell volume. Other cells accumulated no biopolymer. In addition, the recombinant strain was shown to co-produce ethanol and PHB under anaerobic conditions. These results demonstrate that the technologically important organism S. cerevisiae is capable of accumulating PHB aerobically and anaerobically at levels similar to some bacterial systems. The easily assayed PHB system also creates a convenient means of probing in vivo the presence of intracellular metabolites which could be useful for studying the intermediary metabolism of S. cerevisiae.