Cloning and biochemical characterization of Co(2+)-activated bromoperoxidase-esterase (perhydrolase) from Pseudomonas putida IF-3 strain.
The gene encoding Co(2+)-activated bromoperoxidase (BPO)-esterase (EST), catalyzing the organic acid-assisted bromination of some organic compounds with H2O2 and Br(-) and quite specific hydrolysis of (R)-acetylthioisobutyric acid methyl ester, was cloned from the chromosomal DNA of the Pseudomonas putida IF-3 strain. The bpo-est gene comprises 831 bp and encoded a protein of 30181 Da. The enzyme was expressed at a high level in Escherichia coli and purified to homogeneity by ammonium sulfate fractionation and two-step column chromatographies. The recombinant enzyme required acetic acid, propionic acid, isobutyric acid or n-butyric acid in addition to H2O2 and Br(-) for the brominating reaction and was activated by Co(2+) ions. It catalyzed the bromination of styrene and indene to give the corresponding racemic bromohydrin. Although the enzyme did not release free peracetic acid in the reaction mixture, chemical reaction with peracetic acid could well explain such enzymatic reactions via a peracetic acid intermediate. The results indicated that the enzyme was a novel Co(2+)-activated organic acid-dependent BPO (perhydrolase)-EST, belonging to the non-metal haloperoxidase-hydrolase family.
Itoh
N
N
Biotechnology Research Center, Toyama Prefectural University, Kosugi, Toyama, Japan. itoh@pu-toyama.ac.jp
Kawanami
T
T
Liu
J Q
JQ
Dairi
T
T
Miyakoshi
M
M
Nitta
C
C
Kimoto
Y
Y
D019342:Acetic Acid / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001965:Bromides / Q000378:metabolism; D002087:Butyrates / Q000378:metabolism; D003001:Cloning, Molecular; D003035:Cobalt / Q000494:pharmacology*; D016384:Consensus Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli; D008401:Gas Chromatography-Mass Spectrometry; D005798:Genes, Bacterial; D006861:Hydrogen Peroxide / Q000378:metabolism; D007192:Indenes / Q000378:metabolism; D058610:Isobutyrates; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010463:Peracetic Acid / Q000378:metabolism; D010544:Peroxidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011422:Propionates / Q000378:metabolism; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D020058:Styrene / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D001965:Bromides; D002087:Butyrates; D004269:DNA, Bacterial; D007192:Indenes; D058610:Isobutyrates; D011422:Propionates; D011993:Recombinant Fusion Proteins; D003035:Cobalt; D020058:Styrene; C093581:indene; C020380:isobutyric acid; D006861:Hydrogen Peroxide; D010544:Peroxidases; C427221:cobalt(II)-activated bromoperoxidase-esterase; D010463:Peracetic Acid; C029658:propionic acid; D019342:Acetic Acid