A Broad Spectrum Racemase in KT2440 Plays a Key Role in Amino Acid Catabolism.

The broad-spectrum amino acid racemase (Alr) of KT2440 preferentially interconverts the l- and d-stereoisomers of Lys and Arg. Despite conservation of broad-spectrum racemases among bacteria, little is known regarding their physiological role. Here we explore potential functional roles for Alr in KT2440. We demonstrate through cellular fractionation that Alr enzymatic activity is found in the periplasm, consistent with its putative periplasm targeting sequence. Specific activity of Alr is highest during exponential growth, and this activity corresponds with an increased accumulation of d-Lys in the growth medium. An gene knockout strain (Δ) was generated and used to assess potential roles for the gene in peptidoglycan structure, producing soluble signaling compounds, and amino acid metabolism. The stationary phase peptidoglycan structure did not differ between wild-type and Δ strains, indicating that products resulting from Alr activity are not incorporated into peptidoglycan under these conditions. RNA-seq was used to assess differences in the transcriptome between the wild-type and Δ strains. Genes undergoing differential expression were limited to those involved in amino acid metabolism. The Δ strain exhibited a limited capacity for catabolism of l-Lys and l-Arg as the sole source of carbon and nitrogen. This is consistent with a predicted role for Alr in catabolism of l-Lys by virtue of its ability to convert l-Lys to d-Lys, which is further catabolized through the l-pipecolate pathway. The metabolic profiles here also implicate Alr in catabolism of l-Arg, although the pathway by which d-Arg is further catabolized is not clear at this time. Overall, data presented here describe the primary role of Alr as important for basic amino acid metabolism.