Biosynthetic alr alanine racemase from Salmonella typhimurium: DNA and protein sequence determination.

The nucleotide sequence of the alr gene encoding the biosynthetic alanine racemase in Salmonella typhimurium is reported. The sequence was determined by the dideoxy chain termination method of Sanger mostly from recombinants derived from shotgun and specific subcloning of a 2.6-kilobase region containing the a h gene. The final bridging of nonoverlapping contiguous sequences was accomplished with the use of synthetic site-specific primers. The alr gene was found to be 1077 base pairs in length encoding a protein of 359 amino acid residues. Comparison of alr with the dadB gene encoding the catabolic alanine racemase in S . typhimurium revealed almost identical size (1077 vs. 1068 base pairs) and 52% sequence identity. The respective gene products displayed 43% homology, which includes a decapeptide bearing the pyridoxal 5'-phosphate binding site. A key building block in the peptidoglycan layer of bacterial cell walls is D-alanine. The biosynthetic route to D-ala'nine is by racemization of L-alanine. Recent studies from these laboratories (Wasserman et al., 1983) demonstrated the presence of two genes, dadB and alr, from Salmonella typhimurium, both encoding alanine racemases. The dadB gene encodes an alanine racemase that maps adjacent to the dadA gene, which in turn encodes a D-alanine dehydrogenase. The dadB and dadA gene products permit Financial support from the National Science Foundation (Grant *Address correspondence to this author at the Department of ChemPCM 8308969) is gratefully acknowledged. istry. Salmonella to grow on L-alanine as a source of carbon and nitrogen, and thus the dadB alanine racemase may have primarily a catabolic function. The dadB gene has been sequenced, the encoded alanine racemase purified to homogeneity (Wasserman et al., 1984), and its molecular basis of susceptibility to P-haloalanine antibacterials determined (Badet et al., 1984). The second alanine racemase gene in S. typhimurium is alr, which has now been mapped to minute 91 and isolated from a X library (E. Daub et al., unpublished results). In the accompanying paper (Esaki & Walsh, 1986), we describe the purification of the cloned alr-encoded alanine racemase to homogeneity, its enzymatic characteristics and susceptibility to haloalanines, and the N-terminal and active site protein 0006-2960/86/0425-3255$01.50/0