The structural gene for AMP nucleosidase. Mapping, cloning, and overproduction of the enzyme.

Abstract

A mutant of Escherichia coli which contained no detectable AMP nucleosidase activity (EC 3.2.2.4) was produced by treatment with nitrosoguanidine and identified by a colorimetric assay for AMP nucleosidase in individual colonies from agar plates. Conjugation experiments indicated a close linkage between the AMP nucleosidase locus (amn) and his. Assays for AMP nucleosidase in E. coli strains with deletions in the his region established that amn is not located between attP2H and mgl . Transduction experiments with bacteriophage P1 were used to construct a linkage map in the his and amn region of the 100-min chromosome map of E. coli, which places amn at 43.3 min. A Rec- strain of E. coli ( FP4102 ) which carries the uvrC to his region (42.1-44.1 min) as an F' episome was used to confirm this location. The episome from FP4102 was used as the source of DNA for cloning amn. BamHI restriction fragments of the episome were inserted into the homologous site of pBR322 and used to transform the Amn- strain of E. coli to Amn+. The transforming plasmid contained a 9-kb (kilobase) insert. Partial restriction of plasmid with ClaI and religation gave a plasmid with a 6-kb insert which retains the amn gene. Restriction mapping of the plasmid has identified ClaI and PstI sites which appear to be within the amn locus. E. coli (Rec-) which contains the 9-kb plasmid of pBR322 containing amn overproduces AMP nucleosidase when grown in the presence of ampicillin. The specific activity of AMP nucleosidase increases from 0.016 mumol/min/mg of protein to 0.32 mumol/min/mg of protein in extracts of the wild type and the plasmid-bearing strains, respectively. A simple purification procedure yields 10 mg of homogeneous AMP nucleosidase from 25 g of packed cells.