Cloning and characterization of the genes for two distinct cephalosporin acylases from a Pseudomonas strain

Abstract

Pseudomonas sp. strain SE83 converts cephalosporin C and 7,1-(4-carboxybutanamido)cephalosporanic acid (GL-7ACA) to 7-aminocephalosporanic acid (7ACA). A DNA library of this strain was constructed in Escherichia coli and screened for the ability to deacylate GL-7ACA to 7ACA. Apparently, two distinct genes, designated acyI and acyll, were cloned on 4.8-and 6.0-kilobase-pair BglII fragments, respectively. The enzymes encoded by the two genes showed different substrate specificities, and the acyll-encoded enzyme was found to yield 7ACA from cephalosporin C by direct deacylation. Expression of the two genes in E. coli was strongly dependent on a promoter of the vector. The coding regions for acyI and acyIl were localized on the 2.5-and 2.8-kilobase-pair fragments, respectively, by subcloning experiments, and high expression of both genes was obtained by placing them under the control of the lacUV5 promoter. The acyII-encoded enzyme was purified and shown to be composed of two nonidentical subunits with molecular weights of 26,000 and 57,000. Maxicell analysis revealed three acyIl-specific polypeptides, two of which corresponded to the above subunits. The third polypeptide with a molecular weight of 83,000 was suggested to be the precursor of both subunits.Penicillin and cephalosporin acylases (penicillin amidohydrolase EC 3.5.1.11) catalyze the deacylation of acyl side chains of penicillin and cephalosporin derivatives to yield 6-aminopenicillanic acid and 7-aminocephalosporanic acid (7ACA), respectively, key intermediates for semisynthetic 1-lactam antibiotics for medical use. Through exhaustive screening, many kinds of such activities have been detected in a wide variety of microorganisms. To our knowledge, however, enzymatic deacylation of cephalosporin C having D-a-aminoadipic acid moiety in the acyl side chain has not been confirmed despite claims for such an activity by a few patent applications.Recently, we have isolated from soil a strain of Pseudomonas sp., designated SE83, which has an activity to convert cephalosporin C to 7ACA (manuscript in preparation). Preliminary studies revealed that strain SE83 yielded 7,-(4-carboxy-5-oxopentanamido)cephalosporanic acid and 7p-(4-carboxybutanamido)cephalosporanic acid in addition to 7ACA from cephalosporin C. These compounds have been known to be generated from cephalosporin C through oxidative deamination (11) and subsequent decarboxylation in the presence of H202 as illustrated in Fig.

Heterologous Protein Production inAcremonium chrysogenum: Expression of Bacterial Cephalosporin C Acylase and Human Thrombomodulin Genes

“…undergoes maturation in A. chrysogenum. Since the maturation of acylase occurred in E. coli, 10) A. chrysogenum, and S. cerevisiae (data not shown), it is suggested that this acylase may be matured by an autocatalytic mechanism. Acylase can directly convert cephalosporin C to 7 ACA.…”

“…10 ) We found this enzyme, and further studies have been made of its properties, moJecular cloning, and gene expression in Escherichia coli. 10) Thrombomodulin (TM), a thrombin receptor on the endothelial cell surface, inhibits several procoagulant functions of thrombin 18.19) and acts as a protein cofactor in thrombin-catalyzed activation of protein C. 20 ) Activated protein C strongly inhibits activation of the blood coagulation pathway. 21-23) Thus, TM converts procoagulant thrombin to an anticoagulant and plays a central role in the protein C anticoagulant pathway.…”

Comparative Whole-Genome Hybridization Reveals Genomic Islands inBrucellaSpecies

“…B. suis and B. abortus genome sequences contain these two cephalosporin acylases encoding ORFs from 16M (317 and 466 amino acids) fused as a single ORF encoding a 761-amino-acid protein 47% identical to 774-amino-acid protein from Pseudomonas species (30). Cephalosporin acylases are involved in the activation of cephalosporin antibiotics, and the eightfold-higher MIC observed for B. ovis relative to B. abortus (49) may be due to lack of acylase from B. ovis.…”

Cloning and nucleotide sequencing of a novel 7 beta-(4-carboxybutanamido)cephalosporanic acid acylase gene of Bacillus laterosporus and its expression in Escherichia coli and Bacillus subtilis

“…As far as we know, this is the first report on a cephalosporin acylase which consists of a single peptide. This feature of Ji acylase was striking to us since it has become accepted that all the enzymes capable of deacylating cephalosporins consist of two heterologous subunits, as were the cases of the GK16 glutaryl 7-ACA acylase (16) and the SE83 cephalosporin C acylase (17). This common feature, shared also by the penicillin G acylases, was certified to apply to not only the acylases from gram-negative strains but also that from a gram-positive strain, as was shown for the Arthrobacter viscosus penicillin G acylase (18).…”