Organization and expression of the Escherichia coli K-12 dad operon encoding the smaller subunit of D-amino acid dehydrogenase and the catabolic alanine racemase

Abstract

A fragment of the Escherichia coli K-12 chromosome complementing the D-amino acid dehydrogenase and catabolic alanine racemase deficiency of a dad operon deletion mutant was cloned in a mini-Mu plasmid. The dadA and dadA genes were localized to a 3.5-kb part of the plasmid insert. The nucleotide sequence of this fragment revealed two open reading frames encoding 432-and 356-amino-acid-long proteins. We show here that they correspond to the dadA and dadX genes. The dadA gene can encode only the smaller of the two subunits of D-amino acid dehydrogenase. A computer search revealed the presence of a flavin adenine dinucleotide-binding motif in the N-terminal domain of the deduced DadA protein sequence. This is in agreement with biochemical data showing that the D-amino acid dehydrogenase contains flavin adenine dinucleotide in its active center. The predicted dadX gene product appeared to be 85% identical to a dadB-encoded catabolic alanine racemase of SalnoneUla typhimurium. The organization of the dadA and dadX genes confirmed our previous conclusion based on the genetic data (J. Wild, J. Hennig, M. Lobocka, W. Walczak, and T. Klopotowski, Mol. Gen. Genet. 198:315-322, 1985) that these genes form an operon. The main transcription start points of the dad operon were determined by primer extension. They are preceded by a putative Cr70 promoter sequence and two cyclic AMP-cyclic AMP receptor protein (cAMP-CRP) binding sites, one of higher and one of lower affinity to CRP. We propose that the high-affinity site, centered 59.5 bp upstream of the main transcription start point, plays a role in cAMP-CRP-mediated activation of dad operon expression in the absence of glucose.

Arginine racemization by coupled catabolic and anabolic dehydrogenases

“…D-alanine dehydrogenase of E. coli was characterized as a membrane-associated D-amino acid dehydrogenase containing FAD and nonheme iron-sulfur chromophore (22). In collaboration with an alanine racemase, this 2-enzyme couple is encoded in a single operon and is responsible for L-alanine catabolism in E. coli (10) and many other bacteria, including P. aeruginosa (7). In comparison, DauA is not a membrane-associated enzyme, because it remained in the soluble fraction after ultracentrifugation, and it is not required for DL-alanine utilization.…”

“…1). In fact, it has been proposed that L-arginine might be converted into D-arginine via racemization (6), reminiscent of L-alanine utilization through a catabolic alanine racemase and D-alanine dehydrogenase in Escherichia coli and many bacteria (9,10). Existence of an arginine racemase in P. aeruginosa was supported by growth complementation of arginine auxotrophs with D-arginine (6).…”

Characterization of endogenous pyridoxal 5′-phosphate-dependent alanine racemase from Bacillus pseudofirmus OF4

“…The gene organization surrounding the alanine racemase gene was alanine dehydrogenase gene, putative alanine: Na + symporter gene and N-carbamoyl-L-amino acid amidohydrolase gene (data not shown). Genes for alanine dehydrogenase and alanine racemase are located side by side and form an operon (2,26). The catabolic alanine racemases (e.g.…”

“…For example, in E. coli the alr gene is constitutively expressed in the cells for peptidoglycan biosynthesis. The expression of the other gene, dadX, is induced when cells are grown in high concentrations of L-or D-alanine (2,5). The enzyme encoded by the dadX gene catalyzes the conversion of L-alanine to the directly oxidizable D-alanine, which is required in the use of L-alanine as a carbon and energy source (6).…”

“…Alanine racemase is a pyridoxal 5′-phosphate (PLP)-dependent enzyme, catalyzing the interconversion of L-alanine and D-alanine. In some bacteria, such as Escherichia coli and Salmonella, two independent alanine racemase genes have been identified in each microorganism (2)(3)(4). For example, in E. coli the alr gene is constitutively expressed in the cells for peptidoglycan biosynthesis.…”