A NOVEL ARYLESTERASE ACTIVE TOWARD 7-AMINOCEPHALOSPORANIC ACID FROM AGROBACTERIUM-RADIOBACTER IFO-12607 - NUCLEOTIDE-SEQUENCE, GENE-EXPRESSION IN ESCHERICHIA-COLI, AND SITE-DIRECTED MUTAGENESIS

A novel arylesterase from Agrobacterium radiobacter IFO 12607 catalyze s the deacetylation of 7-aminocephalosporanic acid (7-ACA) to form dea cetyl 7-ACA, but is inactive with cephalosporin C. A DNA fragment carr ying the gene encoding the 7-ACA-deacetylating enzyme was cloned from the chromosomal DNA of this bacterium. The open reading frame encoding the enzyme was 642 bp long, corresponding to a protein of 214 amino a cid residues (molecular mass=23,085). The deduced amino acid sequence did not contain the sequence GXSXG, typical of the many serine esteras es including Bacillus cephalosporin C deacetylase, but has the pentape ptide motif sequence GDSLT (amino acid position 9-13) which is also a consensus sequence of some serine esterases. The newly cloned gene was expressed in Escherichia coli under the control of the lac promoter, and the gene product purified from E. coli exhibited the same catalyti c properties as the enzyme purified from A. radiobacter. Site-directed mutagenesis of S11A or S11C within the pentapeptide motif sequence le d to complete loss of the enzyme activity. Thus, the Ser-ll residue wi thin the GDSLT motif sequence was determined to construct the catalyti c center. These results together with those of our previous studies in dicated that the 7-ACA-deacetylating enzyme from A. radiobacter IFO 12 607 is a new member of the family of lipolytic serine esterases contai ning the GDSLT sequence as their catalytic center.