EXISTENCE OF 2 D-ALANINE - D-ALANINE LIGASES IN ESCHERICHIA-COLI - CLONING AND SEQUENCING OF THE DDLA GENE AND PURIFICATION AND CHARACTERIZATION OF THE DDLA AND DDLB ENZYMES

Two distinct genes encoding D-alanine:D-alanine (D-Ala-D-Ala) ligase (ADP forming) activity in Escherichia coli have been cloned by complementation of E. coli strain ST640(lambda-112) deficient in D-Ala-D-Ala ligase activity with a lambda-library of E. coli DNA. One of the two genes, designated as ddlB, is identical with the ddl gene already sequenced [Robinson, A. C., Kenan, D. L., Sweeney, J., & Donachie, W. D. (1986) J. Bacteriol. 167, 809-817]. We describe the subcloning and DNA sequencing of the other gene, designated as ddlA on the basis of similarities with the Salmonella typhimurium ddlA gene [Daub, E., Zawadzke, L. E., Botstein, D., & Walsh, C. T. (1988) Biochemistry 27, 3701-3708]. The predicted amino acid sequence of the E. coli DdlA enzyme shows 90% homology with the S. typhimurium DdlA sequence. The ddlB gene was subcloned by use of the polymerase chain reaction into an expression vector containing an optimized ribosome binding site, which expressed the DdlB enzyme to > 50% soluble cell protein. Both DdlA and DdlB enzymes were purified to > 90% homogeneity and characterized kinetically.