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Characterization of Lactobacillus salivarius alanine racemase: short-chain carboxylate-activation and the role of A131.

Kobayashi J, Yukimoto J, Shimizu Y, Ohmori T, Suzuki H, Doi K, Ohshima T - Springerplus (2015)

Bottom Line: 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.The activity of ALR(A131K) was about three times greater than that of ALR.It thus appears that A131 mediates the activation and stabilization of L. salivarius ALR by short chain carboxylates.

View Article: PubMed Central - PubMed

Affiliation: Microbial Genetics Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan.

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 (ALR(A131K)) substitution was prepared, and its properties were compared with those of ALR. The activity of ALR(A131K) 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), ALR(A131K) 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 ALR(A131K) 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.

No MeSH data available.


Related in: MedlinePlus

Kinetics of acetate inhibition of ALR and ALRA131K. a, b ALR and ALRA131K were assayed for 5 min at 30 °C and pH 7.0 (MES buffer) using various concentrations of l-alanine and acetate as the substrate and inhibitor, respectively. Shown are double reciprocal plots of the initial velocity of ALR (a) or ALRA131K (b) against l-alanine concentrations at several concentrations of acetate (n = 3 for each enzyme). Open circles, without acetate; filled circles, 1.0 mM acetate; open squares, 10.0 mM acetate; filled squares, 20.0 mM acetate; open triangles, 40.0 mM acetate
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Fig4: Kinetics of acetate inhibition of ALR and ALRA131K. a, b ALR and ALRA131K were assayed for 5 min at 30 °C and pH 7.0 (MES buffer) using various concentrations of l-alanine and acetate as the substrate and inhibitor, respectively. Shown are double reciprocal plots of the initial velocity of ALR (a) or ALRA131K (b) against l-alanine concentrations at several concentrations of acetate (n = 3 for each enzyme). Open circles, without acetate; filled circles, 1.0 mM acetate; open squares, 10.0 mM acetate; filled squares, 20.0 mM acetate; open triangles, 40.0 mM acetate

Mentions: The initial velocity of d-alanine formation from l-alanine catalyzed by ALR was measured at several l-alanine concentrations in the presence of five concentrations of acetate. The resultant Lineweaver–Burk plots of the relation between the l-alanine concentration and the initial velocity showed five straight lines (Fig. 4a, b). Although ALR was strongly activated by the addition of 1 mM acetate, the activation level was reduced by the addition of acetate concentrations above 10, and 40 mM acetate greatly inhibited ALR activity at all l-alanine concentrations (Fig. 4a). By contrast, ALRA131K activity was inhibited or unaffected at all the acetate contractions tested (1–40 mM). The Km values of ALR and ALRA131K for l-alanine in absence of acetate were 11.5 and 9.20 mM, respectively. The Vmax of ALR and ALRA131K in absence of acetate were 272 and 751 μmol/min mg, respectively.Fig. 4


Characterization of Lactobacillus salivarius alanine racemase: short-chain carboxylate-activation and the role of A131.

Kobayashi J, Yukimoto J, Shimizu Y, Ohmori T, Suzuki H, Doi K, Ohshima T - Springerplus (2015)

Kinetics of acetate inhibition of ALR and ALRA131K. a, b ALR and ALRA131K were assayed for 5 min at 30 °C and pH 7.0 (MES buffer) using various concentrations of l-alanine and acetate as the substrate and inhibitor, respectively. Shown are double reciprocal plots of the initial velocity of ALR (a) or ALRA131K (b) against l-alanine concentrations at several concentrations of acetate (n = 3 for each enzyme). Open circles, without acetate; filled circles, 1.0 mM acetate; open squares, 10.0 mM acetate; filled squares, 20.0 mM acetate; open triangles, 40.0 mM acetate
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4628008&req=5

Fig4: Kinetics of acetate inhibition of ALR and ALRA131K. a, b ALR and ALRA131K were assayed for 5 min at 30 °C and pH 7.0 (MES buffer) using various concentrations of l-alanine and acetate as the substrate and inhibitor, respectively. Shown are double reciprocal plots of the initial velocity of ALR (a) or ALRA131K (b) against l-alanine concentrations at several concentrations of acetate (n = 3 for each enzyme). Open circles, without acetate; filled circles, 1.0 mM acetate; open squares, 10.0 mM acetate; filled squares, 20.0 mM acetate; open triangles, 40.0 mM acetate
Mentions: The initial velocity of d-alanine formation from l-alanine catalyzed by ALR was measured at several l-alanine concentrations in the presence of five concentrations of acetate. The resultant Lineweaver–Burk plots of the relation between the l-alanine concentration and the initial velocity showed five straight lines (Fig. 4a, b). Although ALR was strongly activated by the addition of 1 mM acetate, the activation level was reduced by the addition of acetate concentrations above 10, and 40 mM acetate greatly inhibited ALR activity at all l-alanine concentrations (Fig. 4a). By contrast, ALRA131K activity was inhibited or unaffected at all the acetate contractions tested (1–40 mM). The Km values of ALR and ALRA131K for l-alanine in absence of acetate were 11.5 and 9.20 mM, respectively. The Vmax of ALR and ALRA131K in absence of acetate were 272 and 751 μmol/min mg, respectively.Fig. 4

Bottom Line: 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.The activity of ALR(A131K) was about three times greater than that of ALR.It thus appears that A131 mediates the activation and stabilization of L. salivarius ALR by short chain carboxylates.

View Article: PubMed Central - PubMed

Affiliation: Microbial Genetics Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan.

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 (ALR(A131K)) substitution was prepared, and its properties were compared with those of ALR. The activity of ALR(A131K) 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), ALR(A131K) 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 ALR(A131K) 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.

No MeSH data available.


Related in: MedlinePlus