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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

Amino acid sequence alignment of L. salivarius ALR with other ALRs: L. sal, L. salivarius UCC 118; L. fer, L. fermentum ATCC14931; P. put, P. putida YZ-26; B. ant, B. anthracis Sterne 43F2; B. pse, B. pseudofirmus OF4; B. sub, B. subtilis PCI 219; G. ste, G. stearothermophilus IFO 12550. The catalytic residues K40 and Y267, the carboxylate binding residues R138, M314 and D315, and A131 are marked with an open circles, filled circles and a filled star, respectively
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Fig1: Amino acid sequence alignment of L. salivarius ALR with other ALRs: L. sal, L. salivarius UCC 118; L. fer, L. fermentum ATCC14931; P. put, P. putida YZ-26; B. ant, B. anthracis Sterne 43F2; B. pse, B. pseudofirmus OF4; B. sub, B. subtilis PCI 219; G. ste, G. stearothermophilus IFO 12550. The catalytic residues K40 and Y267, the carboxylate binding residues R138, M314 and D315, and A131 are marked with an open circles, filled circles and a filled star, respectively

Mentions: We found a gene homologous (Q1WV14 in Uniprot) with ALR in an L. salivarius DNA database, and compared amino acid compositions of L. salivarius with other sources of ALRs (Table 1). The amino acid composition of the L. salivarius ALR was different from those of other ALRs in contents of arginine and lysine. However, affections to characteristics of L. salivarius ALR by these differences are unclear. Thus we aligned and compared the deduced amino acid sequence with those of ALRs from six other sources (Fig. 1). Although L. salivarius ALR showed relatively low overall sequence homology with ALRs from B. anthracis Sterne 43F2 (35.0 %), B. pseudofirmus OF4 (26.1 %), B. subtilis 168 (34.6 %), G. stearothermophilus IFO 12550 (35.6 %) and P. putida YZ-26 (20.4 %), the two catalytic bases in the active site, K40 and Y267, were strictly conserved (Tanizawa et al. 1988; Shaw et al. 1997; Watanabe et al. 2002). In addition, among the four residues (K129, R138, M314 and D315) reportedly responsible for the binding of carboxylates such as acetate and propionate, which are specific inhibitors in the case of ALRs from Bacillus and Geobacillus species (Morollo et al. 1999; Kanodia et al. 2009), R138, M314 and D315 are conserved in the sequence of L. salivarius ALR. On the other hand, K129 is replaced by A131 in L. salivarius ALR, which suggests carboxylates may exert a different effect on L. salivarius ALR.Table 1


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)

Amino acid sequence alignment of L. salivarius ALR with other ALRs: L. sal, L. salivarius UCC 118; L. fer, L. fermentum ATCC14931; P. put, P. putida YZ-26; B. ant, B. anthracis Sterne 43F2; B. pse, B. pseudofirmus OF4; B. sub, B. subtilis PCI 219; G. ste, G. stearothermophilus IFO 12550. The catalytic residues K40 and Y267, the carboxylate binding residues R138, M314 and D315, and A131 are marked with an open circles, filled circles and a filled star, respectively
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Amino acid sequence alignment of L. salivarius ALR with other ALRs: L. sal, L. salivarius UCC 118; L. fer, L. fermentum ATCC14931; P. put, P. putida YZ-26; B. ant, B. anthracis Sterne 43F2; B. pse, B. pseudofirmus OF4; B. sub, B. subtilis PCI 219; G. ste, G. stearothermophilus IFO 12550. The catalytic residues K40 and Y267, the carboxylate binding residues R138, M314 and D315, and A131 are marked with an open circles, filled circles and a filled star, respectively
Mentions: We found a gene homologous (Q1WV14 in Uniprot) with ALR in an L. salivarius DNA database, and compared amino acid compositions of L. salivarius with other sources of ALRs (Table 1). The amino acid composition of the L. salivarius ALR was different from those of other ALRs in contents of arginine and lysine. However, affections to characteristics of L. salivarius ALR by these differences are unclear. Thus we aligned and compared the deduced amino acid sequence with those of ALRs from six other sources (Fig. 1). Although L. salivarius ALR showed relatively low overall sequence homology with ALRs from B. anthracis Sterne 43F2 (35.0 %), B. pseudofirmus OF4 (26.1 %), B. subtilis 168 (34.6 %), G. stearothermophilus IFO 12550 (35.6 %) and P. putida YZ-26 (20.4 %), the two catalytic bases in the active site, K40 and Y267, were strictly conserved (Tanizawa et al. 1988; Shaw et al. 1997; Watanabe et al. 2002). In addition, among the four residues (K129, R138, M314 and D315) reportedly responsible for the binding of carboxylates such as acetate and propionate, which are specific inhibitors in the case of ALRs from Bacillus and Geobacillus species (Morollo et al. 1999; Kanodia et al. 2009), R138, M314 and D315 are conserved in the sequence of L. salivarius ALR. On the other hand, K129 is replaced by A131 in L. salivarius ALR, which suggests carboxylates may exert a different effect on L. salivarius ALR.Table 1

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