Abstract
Many strains of lactic acid bacteria produce high concentrations of d-amino acids. Among them, Lactobacillus salivarius UCC 118 produces d-alanine at a relative concentration much greater than 50 % of the total d, l-alanine (100d/d, l-alanine). We characterized the L. salivarius alanine racemase (ALR) likely responsible for this d-alanine production and found that the enzyme was activated by carboxylates, which is an unique characteristic among ALRs. In addition, alignment of the amino acid sequences of several ALRs revealed that A131 of L. salivarius ALR is likely involved in the activation. To confirm that finding, an L. salivarius ALR variant with an A131K (ALRA131K) substitution was prepared, and its properties were compared with those of ALR. The activity of ALRA131K was about three times greater than that of ALR. In addition, whereas L. salivarius ALR was strongly activated by low concentrations (e.g., 1 mM) of short chain carboxylates, and was inhibited at higher concentrations (e.g., 10 mM), ALRA131K was clearly inhibited at all carboxylate concentrations tested (1–40 mM). Acetate also increased the stability of ALR such that maximum activity was observed at 35 °C and pH 8.0 without acetate, but at 50 °C in the presence of 1 mM acetate. On the other hand, maximum ALRA131K activity was observed at 45 °C and around pH 9.0 with or without acetate. It thus appears that A131 mediates the activation and stabilization of L. salivarius ALR by short chain carboxylates.
Highlights
With the exception for glycine, which contains no asymmetric carbon, all proteogenic amino acids exist as l-αform molecules
Up to around 1980, d-amino acids as enantiomers of corresponding l-amino acids were generally considered to have no important role in living organisms, and so attracted little attention, though it was acknowledged that some d-amino acids were among the main constituents of bacterial cell walls (Heijenoort 2001)
We found a gene homologous (Q1WV14 in Uniprot) with alanine racemase (ALR) in an L. salivarius DNA database, and compared amino acid compositions of L. salivarius with other sources of ALRs (Table 1)
Summary
With the exception for glycine, which contains no asymmetric carbon, all proteogenic amino acids exist as l-αform molecules. Up to around 1980, d-amino acids as enantiomers of corresponding l-amino acids were generally considered to have no important role in living organisms, and so attracted little attention, though it was acknowledged that some d-amino acids were among the main constituents of bacterial cell walls (Heijenoort 2001). It was recently reported that d-amino acids exert inhibitory effects on biofilm formation (Kolodkin-Gal et al 2010; Hochbaum et al.2011). Such evidence of the physiological importance of d-amino acids has prompted investigation into their metabolism and the enzymes involved. Our group recently found that many lactic acid bacteria secrete d-alanine into their medium (Mutaguchi et al 2013) and the relative percentage (100d/(d + l)) of d-alanine per total d, l-alanine in many lactic acid bacterial cells is more than 50 %; the value for Lactobacillus salivarius is 89.6 %, for example
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