Contains also alpha-amino acid ester hydrolase (Beta-Lactam Acylase), glutaryl 7-aca acylases, antibiotic hydrolase, diester hydrolase. Able to perform both hydrolysis and synthesis of a variety of -amino -lactam antibiotics including (R)-ampicillin and cephalexin. The closest relatives is the S15 X-prolyl amino peptidase of Lactobacillus
alpha-Amino ester hydrolases (AEH) are a small class of proteins, which are highly specific for hydrolysis or synthesis of alpha-amino containing amides and esters including beta-lactam antibiotics such as ampicillin, amoxicillin, and cephalexin. A BLAST search revealed the sequence of a putative glutaryl 7-aminocephalosporanic acid (GL-7-ACA) acylase 93% identical to a known AEH from Xanthomonas citri. The gene, termed gaa, was cloned from the genomic DNA of Xanthomonas campestris pv. campestris sp. strain ATCC 33913 and the corresponding protein was expressed into Escherichia coli. The purified protein was able to perform both hydrolysis and synthesis of a variety of alpha-amino beta-lactam antibiotics including (R)-ampicillin and cephalexin, with optimal ampicillin hydrolytic activity at 25 degrees C and pH 6.8, with kinetic parameters of k(cat) of 72.5 s(-1) and K(M) of 1.1 mM. The synthesis parameters alpha, beta(o), and gamma for ampicillin, determined here first for this class of proteins, are alpha = 0.25, beta(o) = 42.8 M(-1), and gamma = 0.23, and demonstrate the excellent synthetic potential of these enzymes. An extensive study of site-directed mutations around the binding pocket of X. campestris pv. campestris AEH strongly suggests that mutation of almost any first-shell amino acid residues around the active site leads to inactive enzyme, including Y82, Y175, D207, D208, W209, Y222, and E309, in addition to those residues forming the catalytic triad, S174, H340, and D307.
Title: Gene cloning and nucleotide sequencing and properties of a cocaine esterase from Rhodococcus sp. strain MB1 Bresler MM, Rosser SJ, Basran A, Bruce NC Ref: Applied Environmental Microbiology, 66:904, 2000 : PubMed
A strain of Rhodococcus designated MB1, which was capable of utilizing cocaine as a sole source of carbon and nitrogen for growth, was isolated from rhizosphere soil of the tropane alkaloid-producing plant Erythroxylum coca. A cocaine esterase was found to initiate degradation of cocaine, which was hydrolyzed to ecgonine methyl ester and benzoate; both of these esterolytic products were further metabolized by Rhodococcus sp. strain MB1. The structural gene encoding a cocaine esterase, designated cocE, was cloned from Rhodococcus sp. strain MB1 genomic libraries by screening recombinant strains of Rhodococcus erythropolis CW25 for growth on cocaine. The nucleotide sequence of cocE corresponded to an open reading frame of 1,724 bp that codes for a protein of 574 amino acids. The amino acid sequence of cocaine esterase has a region of similarity with the active serine consensus of X-prolyl dipeptidyl aminopeptidases, suggesting that the cocaine esterase is a serine esterase. The cocE coding sequence was subcloned into the pCFX1 expression plasmid and expressed in Escherichia coli. The recombinant cocaine esterase was purified to apparent homogeneity and was found to be monomeric, with an M(r) of approximately 65,000. The apparent K(m) of the enzyme (mean +/- standard deviation) for cocaine was measured as 1.33 +/- 0.085 mM. These findings are of potential use in the development of a linked assay for the detection of illicit cocaine.
