IDENTIFICATION, SEQUENCING AND MUTAGENESIS OF THE GENE FOR A D-CARBAMOYLASE FROM AGROBACTERIUM-RADIOBACTER

A clone positive for D-carbamoylase activity (2.7 kb HindIII-BamHI DNA fragment) was obtained by screening a genomic library of Agrobacteriu m radiobacter in Escherichia coli. This DNA fragment contains an open reading frame of 912 bp which is predicted to encode a peptide of 304 amino acids with a calculated molecular mass of 34247 Da. The D-carbam oylase gene, named cauA, was placed under the control of T7 RNA-depend ent promoter and expressed in E. coli BL21(DE3). After induction with isopropyl-thio-beta-o-galactopyranoside, the synthesis of D-carbamoyla se in E. coli reached about 40% of the total protein. The expressed pr otein was shown to possess a molecular mass, on SDS-PAGE, of 36 kDa an d showed an enhanced stability with respect to that of the wild-type e nzyme derived from A. radiobacter. Site-directed mutagenesis experimen ts allowed us to establish that a pro(14)-->Leu(14) exchange leads to an inactive enzyme species, while a Cys(279)-->Ser(279) exchange did n ot impair the functional properties of the enzyme.