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Identification and biochemical evidence of a medium-chain-length polyhydroxyalkanoate depolymerase in the Bdellovibrio bacteriovorus predatory hydrolytic arsenal.

ABSTRACT: The obligate predator Bdellovibrio bacteriovorus HD100 shows a large set of proteases and other hydrolases as part of its hydrolytic arsenal needed for its predatory life cycle. We present genetic and biochemical evidence that open reading frame (ORF) Bd3709 of B. bacteriovorus HD100 encodes a novel medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase (PhaZ(Bd)). The primary structure of PhaZ(Bd) suggests that this enzyme belongs to the ?/?-hydrolase fold family and has a typical serine hydrolase catalytic triad (serine-histidine-aspartic acid) in agreement with other PHA depolymerases and lipases. PhaZ(Bd) has been extracellularly produced using different hypersecretor Tol-pal mutants of Escherichia coli and Pseudomonas putida as recombinant hosts. The recombinant PhaZ(Bd) has been characterized, and its biochemical properties have been compared to those of other PHA depolymerases. The enzyme behaves as a serine hydrolase that is inhibited by phenylmethylsulfonyl fluoride. It is also affected by the reducing agent dithiothreitol and nonionic detergents like Tween 80. PhaZ(Bd) is an endoexohydrolase that cleaves both large and small PHA molecules, producing mainly dimers but also monomers and trimers. The enzyme specifically degrades mcl-PHA and is inactive toward short-chain-length polyhydroxyalkanoates (scl-PHA) like polyhydroxybutyrate (PHB). These studies shed light on the potentiality of these predators as sources of new biocatalysts, such as an mcl-PHA depolymerase, for the production of enantiopure hydroxyalkanoic acids and oligomers as building blocks for the synthesis of biobased polymers.

SUBMITTER: Martinez V

PROVIDER: S-EPMC3416617 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Identification and biochemical evidence of a medium-chain-length polyhydroxyalkanoate depolymerase in the Bdellovibrio bacteriovorus predatory hydrolytic arsenal.

Martínez Virginia V Martínez Virginia V de la Peña Fernando F García-Hidalgo Javier J de la Mata Isabel I García José Luis JL Prieto María Auxiliadora MA

Applied and environmental microbiology 20120615 17

The obligate predator Bdellovibrio bacteriovorus HD100 shows a large set of proteases and other hydrolases as part of its hydrolytic arsenal needed for its predatory life cycle. We present genetic and biochemical evidence that open reading frame (ORF) Bd3709 of B. bacteriovorus HD100 encodes a novel medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase (PhaZ(Bd)). The primary structure of PhaZ(Bd) suggests that this enzyme belongs to the α/β-hydrolase fold family and has a typical seri ...[more]

PMID: 22706067

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Project description:Over the course of the last 3 decades the role of the second messenger cyclic di-GMP (c-di-GMP) as a master regulator of bacterial physiology was determined. Although the control over c-di-GMP levels via synthesis and breakdown and the allosteric regulation of c-di-GMP over receptor proteins (effectors) and riboswitches have been extensively studied, relatively few effectors have been identified and most are of unknown functions. The obligate predatory bacterium Bdellovibrio bacteriovorus has a peculiar dimorphic life cycle, in which a phenotypic transition from a free-living attack phase (AP) to a sessile, intracellular predatory growth phase (GP) is tightly regulated by specific c-di-GMP diguanylate cyclases. B. bacteriovorus also bears one of the largest complement of defined effectors, almost none of known functions, suggesting that additional proteins may be involved in c-di-GMP signaling. In order to uncover novel c-di-GMP effectors, a c-di-GMP capture-compound mass-spectroscopy experiment was performed on wild-type AP and host-independent (HI) mutant cultures, the latter serving as a proxy for wild-type GP cells. Eighty-four proteins were identified as candidate c-di-GMP binders. Of these proteins, 65 did not include any recognized c-di-GMP binding site, and 3 carried known unorthodox binding sites. Putative functions could be assigned to 59 proteins. These proteins are included in metabolic pathways, regulatory circuits, cell transport, and motility, thereby creating a potentially large c-di-GMP network. False candidate effectors may include members of protein complexes, as well as proteins binding nucleotides or other cofactors that were, respectively, carried over or unspecifically interacted with the capture compound during the pulldown. Of the 84 candidates, 62 were found to specifically bind the c-di-GMP capture compound in AP or in HI cultures, suggesting c-di-GMP control over the whole-cell cycle of the bacterium. High affinity and specificity to c-di-GMP binding were confirmed using microscale thermophoresis with a hypothetical protein bearing a PilZ domain, an acyl coenzyme A dehydrogenase, and a two-component system response regulator, indicating that additional c-di-GMP binding candidates may be bona fide novel effectors.In this study, 84 putative c-di-GMP binding proteins were identified in B. bacteriovorus, an obligate predatory bacterium whose lifestyle and reproduction are dependent on c-di-GMP signaling, using a c-di-GMP capture compound precipitation approach. This predicted complement covers metabolic, energy, transport, motility and regulatory pathways, and most of it is phase specific, i.e., 62 candidates bind the capture compound at defined modes of B. bacteriovorus lifestyle. Three of the putative binders further demonstrated specificity and high affinity to c-di-GMP via microscale thermophoresis, lending support for the presence of additional bona fide c-di-GMP effectors among the pulled-down protein repertoire.

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Dataset Information

Identification and biochemical evidence of a medium-chain-length polyhydroxyalkanoate depolymerase in the Bdellovibrio bacteriovorus predatory hydrolytic arsenal.

Publications

Identification and biochemical evidence of a medium-chain-length polyhydroxyalkanoate depolymerase in the Bdellovibrio bacteriovorus predatory hydrolytic arsenal.

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