title: Purification and characterization of YxeI, a penicillin acylase from Bacillus subtilis.
abstract: The paper reports the purification and characterization of the first penicillin acylase from Bacillus subtilis. YxeI, the protein annotated as hypothetical, coded by the gene yxeI in the open reading frame between iol and hut operons in B. subtilis was cloned and expressed in Eshcherichia coli, purified and characterized. The purified protein showed measurable penicillin acylase activity with penicillin V. The enzyme was a homotetramer of 148 kDa. The apparent K(m) of the enzyme for penicillin V and the synthetic substrate 2-nitro-5-(phenoxyacetamido)-benzoic acid was 40 mM and 0.63 mM, respectively, and the association constants were 8.93×10(2) M(-1) and 2.51×10(5) M(-1), respectively. It was inhibited by cephalosporins and conjugated bile salts, substrates of the closely related bile acid hydrolases. It had good sequence homology with other penicillin V acylases and conjugated bile acid hydrolases, members of the Ntn hydrolase family. The N-terminal nucleophile was a cysteine which is revealed by a simple removal of N-formyl-methionine. The activity of the protein was affected by high temperature, acidic pH and the presence of the denaturant guanidine hydrochloride.
authors: Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
authors: Priya
authors: P
authors: Rathinaswamy
authors: Sushama M
authors: SM
authors: Gaikwad
authors: C G
authors: CG
authors: Suresh
authors: Asmita A
authors: AA
authors: Prabhune
authors: James A
authors: JA
authors: Brannigan
authors: Guy G
authors: GG
authors: Dodson
authors: Archana V
authors: AV
authors: Pundle
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001647:Bile Acids and Salts / Q000494:pharmacology; D002511:Cephalosporins / Q000494:pharmacology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D019791:Guanidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010399:Penicillin Amidase / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D001647:Bile Acids and Salts; D002511:Cephalosporins; D010399:Penicillin Amidase; D019791:Guanidine