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1.
Kazunori Ushimaru Yoko Motoda Keiji Numata Takeharu Tsuge 《Applied and environmental microbiology》2014,80(9):2867-2873
In this study, we performed in vitro and in vivo activity assays of polyhydroxyalkanoate (PHA) synthases (PhaCs) in the presence of phasin proteins (PhaPs), which revealed that PhaPs are activators of PhaC derived from Aeromonas caviae (PhaCAc). In in vitro assays, among the three PhaCs tested, PhaCAc was significantly activated when PhaPs were added at the beginning of polymerization (prepolymerization PhaCAc), whereas the prepolymerization PhaCRe (derived from Ralstonia eutropha) and PhaCDa (Delftia acidovorans) showed reduced activity with PhaPs. The PhaP-activated PhaCAc showed a slight shift of substrate preference toward 3-hydroxyhexanoyl-CoA (C6). PhaPAc also activated PhaCAc when it was added during polymerization (polymer-elongating PhaCAc), while this effect was not observed for PhaCRe. In an in vivo assay using Escherichia coli TOP10 as the host strain, the effect of PhaPAc expression on PHA synthesis by PhaCAc or PhaCRe was examined. As PhaPAc expression increased, PHA production was increased by up to 2.3-fold in the PhaCAc-expressing strain, whereas it was slightly increased in the PhaCRe-expressing strain. Taken together, this study provides evidence that PhaPs function as activators for PhaCAc both in vitro and in vivo but do not activate PhaCRe. This activating effect may be attributed to the new role of PhaPs in the polymerization reaction by PhaCAc. 相似文献
2.
By in vitro evolution experiment, we have first succeeded in acquiring higher active mutants of a synthase that is a key enzyme essential for bacterial synthesis of biodegradable polyester, polyhydroxyalkanoate (PHA). Aeromonas caviae FA440 synthase, termed PhaC(Ac), was chosen as a good target for evolution, since it can synthesize a PHA random copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate [P(3HB-co-3HHx)] that is a tough and flexible material compared to polyhydroxybutyrate (PHB) homopolyester. The in vitro enzyme evolution system consists of PCR-mediated random mutagenesis targeted to a limited region of the phaC(Ac) gene and screening mutant enzymes with higher activities based on two types of polyester accumulation system by using Escherichia coli for the synthesis of PHB (by JM109 strain) (S. Taguchi, A. Maehara, K. Takase, M. Nakahara, H. Nakamura, and Y. Doi, FEMS Microbiol. Lett. 198:65-71, 2001) and of P(3HB-co-3HHx) [by LS5218 [fadR601 atoC(Con)] strain]. The expression vector for the phaC(Ac) gene, together with monomer-supplying enzyme genes, was designed to synthesize PHB homopolyester from glucose and P(3HB-co-3HHx) copolyester from dodecanoate. Two evolved mutant enzymes, termed E2-50 and T3-11, screened through the evolution system exhibited 56 and 21% increases in activity toward 3HB-coenzyme A, respectively, and consequently led to enhanced accumulation (up to 6.5-fold content) of P(3HB-co-3HHx) in the recombinant LS5218 strains. Two single mutations in the mutants, N149S for E2-50 and D171G for T3-11, occurred at positions that are not highly conserved among the PHA synthase family. It should be noted that increases in the 3HHx fraction (up to 16 to 18 mol%) were observed for both mutants compared to the wild type (10 mol%). 相似文献
3.
Expression and Characterization of (R)-Specific Enoyl Coenzyme A Hydratase Involved in Polyhydroxyalkanoate Biosynthesis by Aeromonas caviae 总被引:2,自引:0,他引:2
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Complementation analysis of a polyhydroxyalkanoate (PHA)-negative mutant of Aeromonas caviae proved that ORF3 in the pha locus (a 402-bp gene located downstream of the PHA synthase gene) participates in PHA biosynthesis on alkanoic acids, and the ORF3 gene is here referred to as phaJAc. Escherichia coli BL21(DE3) carrying phaJAc under the control of the T7 promoter overexpressed enoyl coenzyme A (enoyl-CoA) hydratase, which was purified by one-step anion-exchange chromatography. The N-terminal amino acid sequence of the purified hydratase corresponded to the amino acid sequence deduced from the nucleotide sequence of phaJAc except for the initial Met residue. The enoyl-CoA hydratase encoded by phaJAc exhibited (R)-specific hydration activity toward trans-2-enoyl-CoA with four to six carbon atoms. These results have demonstrated that (R)-specific hydration of 2-enoyl-CoA catalyzed by the translated product of phaJAc is a channeling pathway for supplying (R)-3-hydroxyacyl-CoA monomer units from fatty acid β-oxidation to poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis in A. caviae. 相似文献
4.
Pantazaki AA Tambaka MG Langlois V Guerin P Kyriakidis DA 《Molecular and cellular biochemistry》2003,254(1-2):173-183
The biosynthesis of polyhydroxyalkanoates (PHAs) was studied, for the first time, in the thermophilic bacterium Thermus thermophilus. Using sodium gluconate (1.5% w/v) or sodium octanoate (10 mM) as sole carbon sources, PHAs were accumulated to approximately 35 or 40% of the cellular dry weight, respectively. Gas chromatographic analysis of PHA isolated from gluconate-grown cells showed that the polyester (Mw: 480,000 g.mol–1) was mainly composed of 3-hydroxydecanoate (3HD) with a molar fraction of 64%. In addition, 3-hydroxyoctanoate (3HO), 3-hydroxyvalerate (3HV) and 3-hydroxybutyrate (3HB) occurred as constituents. In contrast, the polyester (Mw: 391,000 g mol–1) from octanoate-grown cells was composed of 24.5 mol% 3HB, 5.4 mol% 3HO, 12.3 mol% 3-hydroxynonanoate (3HN), 14.6 mol% 3HD, 35.4 mol% 3-hydroxyundecanoate (3HUD) and 7.8 mol% 3-hydroxydodecanoate (3HDD). Activities of PHA synthase, a -ketothiolase and an NADPH-dependent reductase were detected in the soluble cytosolic fraction obtained from gluconate-grown cells of T. thermophilus. The soluble PHA synthase was purified 4271-fold with 8.5% recovery from gluconate-grown cells, presenting a Km of 0.25 mM for 3HB-CoA. The optimal temperature of PHA synthase activity was about 70°C and acts optimally at pH near 7.3. PHA synthase activity was inhibited 50% with 25 M CoA and lost all of its activity when it was treated with alkaline phosphatase. PHA synthase, in contrary to other reported PHA synthases did not exhibit a lag phase on its kinetics, when low concentration of the enzyme was used. Incubation of PHA synthase with 1 mM N-ethyl-maleimide inhibits the enzyme 56%, indicating that cysteine might be involved in the catalytic site of the enzyme. Acetyl phosphate (10 mM) activated both the native and the dephosphorylated enzyme. A major protein (55 kDa) was detected by SDS-PAGE. When a partially purified preparation was analyzed on native PAGE the major band exhibiting PHA synthase activity was eluted from the gel and analyzed further on SDS-PAGE, presenting the first purification of a PHA synthase from a thermophilic microorganism. 相似文献
5.
This study investigated the apparent genetic redundancy in the biosynthesis of polyhydroxyalkanoates (PHAs) in the Rhodospirillum rubrum genome revealed by the occurrence of three homologous PHA polymerase genes (phaC1, phaC2, and phaC3). In vitro biochemical assays established that each gene product encodes PHA polymerase. A series of single, double, and triple phaC deletion mutants were characterized with respect to PHA production and growth capabilities on acetate or hexanoate as the sole carbon source. These analyses establish that phaC2 contributes the major capacity to produce PHA, even though the PhaC2 protein is not the most efficient PHA polymerase biocatalyst. In contrast, phaC3 is an insignificant contributor to PHA productivity, and phaC1, the PHA polymerase situated in the PHA biosynthetic operon, plays a minor role in this capability, even though both of these genes encode PHA polymerases that are more efficient enzymes. These observations are consistent with the finding that PhaC1 and PhaC3 occur at undetectable levels, at least 10-fold lower than that of PhaC2. The monomers in the PHA polymer produced by these strains establish that PhaC2 is responsible for the incorporation of the C5 and C6 monomers. The in vitro characterizations indicate that heteromeric PHA polymerases composed of mixtures of different PhaC paralogs are more efficient catalysts, suggesting that these proteins form complexes. Finally, the physiological role of PHA accumulation in enhancing the fitness of R. rubrum was indicated by the relationship between PHA content and growth capabilities of the genetically manipulated strains that express different levels of the PHA polymer. 相似文献
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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. 相似文献
8.
Jo-Ann Chuah Satoshi Tomizawa Miwa Yamada Takeharu Tsuge Yoshiharu Doi Kumar Sudesh Keiji Numata 《Applied and environmental microbiology》2013,79(12):3813-3821
Saturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase from Chromobacterium sp. strain USM2 (PhaCCs) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaCCs for 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity. In vitro activities for polymerization of 3HV and 3HHx monomers were consistent with in vivo substrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C4 and C5) or MCL (C6) substrates substantiates the fundamental classification of PHA synthases. 相似文献
9.
Coexpression of Genetically Engineered 3-Ketoacyl-ACP Synthase III (fabH) and Polyhydroxyalkanoate Synthase (phaC) Genes Leads to Short-Chain-Length-Medium-Chain-Length Polyhydroxyalkanoate Copolymer Production from Glucose in Escherichia coli JM109
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Christopher T. Nomura Kazunori Taguchi Seiichi Taguchi Yoshiharu Doi 《Applied microbiology》2004,70(2):999-1007
Polyhydroxyalkanoates (PHAs) can be divided into three main types based on the sizes of the monomers incorporated into the polymer. Short-chain-length (SCL) PHAs consist of monomer units of C3 to C5, medium-chain-length (MCL) PHAs consist of monomer units of C6 to C14, and SCL-MCL PHAs consist of monomers ranging in size from C4 to C14. Although previous studies using recombinant Escherichia coli have shown that either SCL or MCL PHA polymers could be produced from glucose, this study presents the first evidence that an SCL-MCL PHA copolymer can be made from glucose in recombinant E. coli. The 3-ketoacyl-acyl carrier protein synthase III gene (fabH) from E. coli was modified by saturation point mutagenesis at the codon encoding amino acid 87 of the FabH protein sequence, and the resulting plasmids were cotransformed with either the pAPAC plasmid, which harbors the Aeromonas caviae PHA synthase gene (phaC), or the pPPAC plasmid, which harbors the Pseudomonas sp. strain 61-3 PHA synthase gene (phaC1), and the abilities of these strains to accumulate PHA from glucose were assessed. It was found that overexpression of several of the mutant fabH genes enabled recombinant E. coli to induce the production of monomers of C4 to C10 and subsequently to produce unusual PHA copolymers containing SCL and MCL units. The results indicate that the composition of PHA copolymers may be controlled by the monomer-supplying enzyme and further reinforce the idea that fatty acid biosynthesis may be used to supply monomers for PHA production. 相似文献
10.
In vivo random mutagenesis of the polyhydroxyalkanoate (PHA) synthase gene from Aeromonas punctata was performed employing the mutator strain Escherichia coli XL1-Red. About 200,000 mutants were screened on Nile red-containing medium and five mutants with enhanced fluorescence were
selected. Four of these mutants exhibited enhanced in vivo and in vitro PHA synthase activity. Mutant M1, which carried the
single mutation F518I, showed a five-fold increase in specific PHA synthase activity, whereas the corresponding mediated PHA
accumulation increased by 20%, as compared with the wild-type PHA synthase. Mutant M2, which carried the single mutation V214G,
showed a two-fold increase in specific PHA synthase activity and PHA accumulation only increased by 7%. Overall, the in vitro
activities of the overproducing mutants ranged from 1.1- to 5-fold more than the wild-type activity, whereas the amounts of
accumulated PHA ranged over 107–126% of that of the wild type. Moreover, all mutants mediated synthesis of PHAs with an increased
weight average molar mass, but the molar fractions of 3-hydroxybutyrate and 3-hydroxyhexanoate remained almost constant. In
vivo random mutagenesis proved to be a versatile tool to isolate mutants exerting improved properties with respect to PHA
biosynthesis.
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11.
Matsumoto K Nagao R Murata T Arai Y Kichise T Nakashita H Taguchi S Shimada H Doi Y 《Biomacromolecules》2005,6(4):2126-2130
In this study, the enhancement of photosynthetic PHA production was achieved using the highly active mutants of PHA synthase created by the in vitro evolutionally techniques. The wild-type and mutated PHA synthase genes from Aeromonas caviae were introduced into Arabidopsis thaliana together with the NADPH-dependent acetoacetyl-CoA reductase gene from Ralstonia eutropha. Expression of the highly active mutated PHA synthase genes, N149S and D171G, led to an 8-10-fold increase in PHA content in the T1 transgenic Arabidopsis, compared to plants harboring the wild-type PHA synthase gene. In homozygous T2 progenies, PHA content was further increased up to 6.1 mg/g cell dry weight. GC/MS analysis of the purified PHA from the transformants revealed that these PHAs were poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymers consisting of 0.2-0.8 mol % 3HV. The monomer composition of the P(3HB-co-3HV) copolymers synthesized by the wild-type and mutated PHA synthases reflected the substrate specificities observed in Escherichia coli. These results indicate that in vitro evolved PHA synthases can enhance the productivity of PHA and regulate the monomer composition in transgenic plants. 相似文献
12.
Jon Romero-Aguirregomezcorta ángela Patricia Santa Francisco Alberto García-Vázquez Pilar Coy Carmen Matás 《PloS one》2014,9(12)
Nitric oxide (NO) is a molecule involved in many reproductive processes. Its importance during oocyte in vitro maturation (IVM) has been demonstrated in various species although sometimes with contradictory results. The objective of this study was to determine the effect of NO during IVM of cumulus oocyte complexes and its subsequent impact on gamete interaction in porcine species. For this purpose, IVM media were supplemented with three NOS inhibitors: NG-nitro-L-arginine methyl ester (L-NAME), NG-monomethyl-L-arginine (L-NMMA) and aminoguanidine (AG). A NO donor, S-nitrosoglutathione (GSNO), was also used. The effects on the cumulus cell expansion, meiotic resumption, zona pellucida digestion time (ZPdt) and, finally, on in vitro fertilization (IVF) parameters were evaluated. The oocyte S-nitrosoproteins were also studied by in situ nitrosylation. The results showed that after 42 h of IVM, AG, L-NAME and L-NMMA had an inhibitory effect on cumulus cell expansion. Meiotic resumption was suppressed only when AG was added, with 78.7% of the oocytes arrested at the germinal vesicle state (P<0.05). Supplementation of the IVM medium with NOS inhibitors or NO donor did not enhance the efficiency of IVF, but revealed the importance of NO in maturation and subsequent fertilization. Furthermore, protein S-nitrosylation is reported for the first time as a pathway through which NO exerts its effect on porcine IVM; therefore, it would be important to determine which proteins are nitrosylated in the oocyte and their functions, in order to throw light on the mechanism of action of NO in oocyte maturation and subsequent fertilization. 相似文献
13.
AIMS: Burkholderia sp. USM (JCM15050) isolated from oil-polluted wastewater is capable of utilizing palm oil products and glycerol to synthesize poly(3-hydroxybutyrate) [P(3HB)]. To confer the ability to produce polymer containing 3-hydroxyhexanoate (3HHx), plasmid (pBBREE32d13) harbouring the polyhydroxyalkanoate (PHA) synthase gene of Aeromonas caviae (phaC(Ac)) was transformed into this strain. Methods and Results: The resulting transformant incorporated approximately 1 ± 0·3 mol% of 3HHx in the polymer when crude palm kernel oil (CPKO) or palm kernel acid oil was used as the sole carbon source. In addition, when the transformed strain was cultivated in the mixtures of CPKO and sodium valerate, PHA containing 69 mol% 3HB, 30 mol% 3-hydroxyvalerate and 1 mol% 3HHx monomers was produced. Batch feeding of carbon sources with 0·5% (v/v) CPKO at 0 h and 0·25% (w/v) sodium valerate at 36 h yielded 6 mol% of 3HHx monomer by controlled-feeding strategies. CONCLUSIONS: Burkholderia sp. USM (JCM15050) has the metabolic pathways to supply both the short-chain length (SCL) and medium-chain length (MCL) PHA monomers. By transforming the strain with the Aer. caviae PHA synthase with broader substrate specificity, SCL-MCL PHA was produced. Significance and Impact of the Study: This is the first study demonstrating the ability of transformant Burkholderia to produce P(3HB-co-3HHx) from a single carbon source. 相似文献
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山茶属CHS基因家族的组成和分子进化初探 总被引:8,自引:0,他引:8
用PCR方法从4种山茶属(Camellia)(山茶科)(Tlaeaceae)植物的总DNA中分别扩增到CHS基因外显子2的部分序列,经克隆、测序得到16个该基因的序列,这些序列与来自GenBank的该属另一种植物的3个序列及作为外类群的大豆(Glycine max (L)Merr)的2个序列一起进行分析。研究表明,山茶属CHS基因家族在进化过程中已分化为A、B、c三个亚家族,包括A1、A2、A3、B1、B2、C等6类不同的基因成员;其中只有A2类成员为全部被研究的5种植物所共有,而其他5类成员只在部分被研究的植物中发现;所有这些CHS成员具有很高的同源性:在核苷酸水平上同一亚家族内基本上高于90%,不同亚家族间也在78%以上。从推测的氨基酸组成看,山茶属内CHS基因的功能已发生了分化,各类成员的碱基替代率有较大差异;从分子系统发育树和可能的氨基酸组成分析,山茶属具有新功能的基因成员是在经过基因重复后,或是由少数几个位点的突变而成,或是由逐渐积累的突变而形成的。进一步分析认为,该属CHS基因的分化直到近期还在活跃地进行,并且不同种的进化式样有一定的差别,这种不同的进化式样可能是物种形成后受不同环境因素影响而形成的。 相似文献
17.
Amastigotes of different strains of Trypanosoma cruzi responded to stimulation with concanavalin A in an axenic medium by increased DNA synthesis and cell multiplication. These effects were inhibited by α-methyl mannoside. Other mitogens, i.e. phytohemagglutinin P, castor bean ricin Type II isolated from Ricinus communis, and a bacterial lipopolysaccharide, had no effect on amastigote growth. Amastigote stimulation by concanavlin A lends itself to studies on the biochemistry and cell cycle of this human pathogen. 相似文献
18.
Jing Han Qiuhe Lu Ligang Zhou Hailong Liu Hua Xiang 《Applied and environmental microbiology》2009,75(19):6168-6175
Genome-wide analysis has revealed abundant FabG (β-ketoacyl-ACP reductase) paralogs, with uncharacterized biological functions, in several halophilic archaea. In this study, we identified for the first time that the fabG1 gene, but not the other five fabG paralogs, encodes the polyhydroxyalkanoate (PHA)-specific acetoacetyl coenzyme A (acetoacetyl-CoA) reductase in Haloarcula hispanica. Although all of the paralogous fabG genes were actively transcribed, only disruption or knockout of fabG1 abolished PHA synthesis, and complementation of the ΔfabG1 mutant with the fabG1 gene restored both PHA synthesis capability and the NADPH-dependent acetoacetyl-CoA reductase activity. In addition, heterologous coexpression of the PHA synthase genes (phaEC) together with fabG1, but not its five paralogs, reconstructed the PHA biosynthetic pathway in Haloferax volcanii, a PHA-defective haloarchaeon. Taken together, our results indicate that FabG1 in H. hispanica, and possibly its counterpart in Haloarcula marismortui, has evolved the distinct function of supplying precursors for PHA biosynthesis, like PhaB in bacteria. Hence, we suggest the renaming of FabG1 in both genomes as PhaB, the PHA-specific acetoacetyl-CoA reductase of halophilic archaea.Several haloarchaeal species belonging to the genera Haloferax, Haloarcula, Natrialba, and Haloquadratum are capable of synthesizing short-chain-length polyhydroxyalkanoates (SCL-PHAs) (6, 8, 14, 16), a large family of biopolymers with desirable biodegradability, biocompatibility, and thermoplastic features (31). Although the metabolic pathways of PHAs in bacteria have been characterized in detail (10, 15, 20, 25, 26, 37), the genes involved in PHA biosynthesis in haloarchaea were not recognized until recently, when the PHA synthase genes were identified and characterized for Haloarcula marismortui, Haloarcula hispanica, and Haloferax mediterranei (6, 19). These archaeal PHA synthases are all composed of two subunits, PhaE and PhaC. They are homologous to the class III PHA synthases from bacteria but have a longer C-terminal extension in the PhaC subunit. Nevertheless, the pathway of supplying the PHA precursors has not yet been clarified for any haloarchaeal strain.Both H. mediterranei and H. hispanica are able to synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from unrelated carbon sources, despite the content of the (R)-3-hydroxyvalerate (3-HV) monomer of PHBV in H. mediterranei (10 to 13 mol%) (4, 19) being much higher than that in H. hispanica (∼3 mol%) (19). Conversely, the bacteria Ralstonia eutropha and Synechocystis sp. strain PCC6803, which possess class I and III PHA synthases, respectively, accumulate just poly(3-hydroxybutyrate) (PHB) when the 3-HV-related carbon sources (i.e., propionate and valerate) are not supplied (30). In these two bacteria, the biosynthesis of the (R)-3-hydroxybutyrate coenzyme A [(R)-3-HB-CoA] precursor is conducted by two steps. First, two acetyl-CoA molecules are condensed into one acetoacetyl-CoA molecule by the enzyme β-ketothiolase (PhaA). The acetoacetyl-CoA is then reduced to (R)-3-HB-CoA by a PHA-specific acetoacetyl-CoA reductase (PhaB). The resulting (R)-3-HB-CoA is subsequently incorporated into PHB, catalyzed by PHA synthases (26, 36).Both PhaB and FabG belong to the short-chain dehydrogenase/reductase (SDR) superfamily, whose members are homologous in sequence and have several conserved motifs (27, 29). Interestingly, although FabGs naturally reduce 3-ketoacyl-ACP to form (R)-3-hydroxyacyl-ACP in fatty acid biosynthesis, a few FabGs also recognize 3-ketoacyl-CoA and hence function in PHA biosynthesis. For example, the FabG proteins of Escherichia coli and Pseudomonas aeruginosa have been demonstrated to supply precursors for PHA biosynthesis in recombinant E. coli cells (21, 22, 32, 35). In addition, several FabG paralogs may have evolved a distinct function, to be responsible only for PHA accumulation. This situation was observed in Synechocystis sp. strain PCC6803, where the originally annotated FabG (12) was renamed PhaB after an understanding of its function in PHA biosynthesis (36).Genome-wide analysis of H. marismortui ATCC 43049 (1) revealed eight FabG paralogs in this haloarchaeon. Similarly, multiple fabG paralog genes (fabG1 to fabG6) were also observed in the newly sequenced genome of H. hispanica (our unpublished data). In this study, we demonstrate that fabG1, but not the other five fabG paralogs, encodes the PHA-specific acetoacetyl-CoA reductase in H. hispanica. It is responsible for providing (R)-3-HB-CoA for PHA biosynthesis in Haloarcula species, and interestingly, this enzyme also functions well in Haloferax volcanii, endowing this PHA-defective strain with the ability to accumulate PHA when cotransformed with PHA synthase genes. 相似文献
19.
Hisano T Tsuge T Fukui T Iwata T Miki K Doi Y 《The Journal of biological chemistry》2003,278(1):617-624
The (R)-specific enoyl coenzyme A hydratase ((R)-hydratase) from Aeromonas caviae catalyzes the addition of a water molecule to trans-2-enoyl coenzyme A (CoA), with a chain-length of 4-6 carbons, to produce the corresponding (R)-3-hydroxyacyl-CoA. It forms a dimer of identical subunits with a molecular weight of about 14,000 and is involved in polyhydroxyalkanoate (PHA) biosynthesis. The crystal structure of the enzyme has been determined at 1.5-A resolution. The structure of the monomer consists of a five-stranded antiparallel beta-sheet and a central alpha-helix, folded into a so-called "hot dog" fold, with an overhanging segment. This overhang contains the conserved residues including the hydratase 2 motif residues. In dimeric form, two beta-sheets are associated to form an extended 10-stranded beta-sheet, and the overhangs obscure the putative active sites at the subunit interface. The active site is located deep within the substrate-binding tunnel, where Asp(31) and His(36) form a catalytic dyad. These residues are catalytically important as confirmed by site-directed mutagenesis and are possibly responsible for the activation of a water molecule and the protonation of a substrate molecule, respectively. Residues such as Leu(65) and Val(130) are situated at the bottom of the substrate-binding tunnel, defining the preference of the enzyme for the chain length of the substrate. These results provide target residues for protein engineering, which will enhance the significance of this enzyme in the production of novel PHA polymers. In addition, this study provides the first structural information of the (R)-hydratase family and may facilitate further functional studies for members of the family. 相似文献
20.
Enhanced PHA stimulation of cancer patients' lymphocytes by addition of BM 12531 (azimexon) in vitro
Summary
Lymphocytes of seven patients with advanced cancer were investigated for changes of blastogenic response to PHA after the addition of the new immunomodulating compound 2-[2-cyanaziridinyl-(1)]-2-[2-carbamoyl-aziridinyl-(1)]-propane (BM 12 531; prop. INN azimexon) to in vitro cultures. Concentrations of 0.2, 1, 2.5, 5, and 10 g/ml were added to cultures. Significant changes in 3H-thymidine uptake were observed with all concentrations of BM 12 531. The most pronounced increase was observed with the concentration of 0.2 g/ml, i.e., five of seven patients had a significantly enhanced blastogenesis response to PHA. Higher doses were effective only in some of the lymphocyte cultures, and stimulation was always lower than with 0.2 g. These observations suggest an interaction of BM 12 531 and lymphocyte activation at a molecular level. 相似文献