A common active site of polyhydroxyalkanoate synthase from Bacillus cereus YB-4 is involved in polymerization and alcoholysis reactions

Abstract

Polyhydroxyalkanoate (PHA) synthase from Bacillus cereus YB-4 (PhaRCYB4) catalyzes not only PHA polymerization but also alcoholytic cleavage of PHA chains. The alcoholysis activity of PhaRCYB4 is expressed when a hydroxyacyl-CoA monomer is absent but an alcohol compound is present. In this study, we performed alanine mutagenesis of the putative catalytic triad (Cys(151), Asp(306), and His(335)) in the PhaCYB4 subunit to identify the active site residues for polymerization and alcoholysis activities. Individual substitution of each triad residue with alanine resulted in loss of both polymerization and alcoholysis activities, suggesting that these residues are commonly shared between polymerization and alcoholysis reactions. The loss of activity was also observed following mutagenesis of the triad to other amino acids, except for one PhaRCYB4 mutant with a C151S substitution, which lost polymerization activity but still possessed cleavage activity towards PHA chains. The low-molecular-weight PHA isolated from the PhaRCYB4(C151S)-expressing strain showed a lower ratio of alcohol capping at the P(3HB) carboxy terminus than did that from the wild-type-expressing strain. This observation implies that hydrolysis activity of PhaRCYB4 might be elicited by the C151S mutation.

Carboxy-terminal modification of polyhydroxyalkanoate (PHA) via alcoholysis reaction catalyzed by Class IV PHA synthase

“…This cysteine is also an active center for polymerization [18]. In the PhaRC YB4 (C151S) mutant, the substituted serine plays a role as an active center for not only alcoholysis activity but also emergent hydrolysis activity [18].…”

Bacillus cereus-type polyhydroxyalkanoate biosynthetic gene cluster contains R-specific enoyl-CoA hydratase gene

“…cereus YB-4 was isolated as a PHA-producing bacterium from ammunition-polluted soil in Kitakyushu City, Japan. 18) In our previous studies, [19][20][21][22][23][24] we demonstrated that the PHA synthase from B. cereus YB-4 (PhaRC YB4 ) catalyzes not only PHA polymerization but also the alcoholytic cleavage of PHA chains. The alcoholysis activity is observed when a PHA monomer is absent but an alcohol compound such as ethanol or propanol is present.…”

Screening Method for Polyhydroxyalkanoate Synthase Mutants Based on Polyester Degree of Polymerization Using High-Performance Liquid Chromatography

“…Poly(3-hydroxybutyrate) (P(3HB)) with an aromatic group at the carboxyl terminal (P(3HB)-93%, number-average molecular weight, M n = 9600, benzyl alcohol terminal modification yield = 93%) was biosynthesized as follows: recombinant E. coli BW25113 ∆adhE (JW1228-KC), an alcohol dehydrogenase deletion strain, harboring pGEM-phaRC YB4 AB (carrying phaRC YB4 from B. cereus YB-4 and a monomer supplying phaAB from Ralstonia eutropha H16) [18] was cultured at 37 • C for 72 h in Luria Bertani (LB) medium (10 g/L sodium chloride [NaCl], 10 g/L tryptone, and 5 g/L yeast extract) supplemented with 20 g/L glucose and 1 mL/L benzyl alcohol. PHAs accumulated in bacterial cells were extracted using chloroform and purified with methanol [19]. The molecular weight was determined by GPC, and the terminal modification yield was calculated from 1 H-NMR spectra [17].…”