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

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 (AL...

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Main Authors:	Kobayashi, Jyumpei, Yukimoto, Jotaro, Shimizu, Yasuhiro, Ohmori, Taketo, Suzuki, Hirokazu, Doi, Katsumi, Ohshima, Toshihisa
Format:	Online
Language:	English
Published:	Springer International Publishing 2015
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4628008/

id	pubmed-4628008
recordtype	oai_dc
spelling	pubmed-46280082015-11-05 Characterization of Lactobacillus salivarius alanine racemase: short-chain carboxylate-activation and the role of A131 Kobayashi, Jyumpei Yukimoto, Jotaro Shimizu, Yasuhiro Ohmori, Taketo Suzuki, Hirokazu Doi, Katsumi Ohshima, Toshihisa Research 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. Springer International Publishing 2015-10-24 /pmc/articles/PMC4628008/ /pubmed/26543773 http://dx.doi.org/10.1186/s40064-015-1335-6 Text en © Kobayashi et al. 2015 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Kobayashi, Jyumpei Yukimoto, Jotaro Shimizu, Yasuhiro Ohmori, Taketo Suzuki, Hirokazu Doi, Katsumi Ohshima, Toshihisa
spellingShingle	Kobayashi, Jyumpei Yukimoto, Jotaro Shimizu, Yasuhiro Ohmori, Taketo Suzuki, Hirokazu Doi, Katsumi Ohshima, Toshihisa Characterization of Lactobacillus salivarius alanine racemase: short-chain carboxylate-activation and the role of A131
author_facet	Kobayashi, Jyumpei Yukimoto, Jotaro Shimizu, Yasuhiro Ohmori, Taketo Suzuki, Hirokazu Doi, Katsumi Ohshima, Toshihisa
author_sort	Kobayashi, Jyumpei
title	Characterization of Lactobacillus salivarius alanine racemase: short-chain carboxylate-activation and the role of A131
title_short	Characterization of Lactobacillus salivarius alanine racemase: short-chain carboxylate-activation and the role of A131
title_full	Characterization of Lactobacillus salivarius alanine racemase: short-chain carboxylate-activation and the role of A131
title_fullStr	Characterization of Lactobacillus salivarius alanine racemase: short-chain carboxylate-activation and the role of A131
title_full_unstemmed	Characterization of Lactobacillus salivarius alanine racemase: short-chain carboxylate-activation and the role of A131
title_sort	characterization of lactobacillus salivarius alanine racemase: short-chain carboxylate-activation and the role of a131
description	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.
publisher	Springer International Publishing
publishDate	2015
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4628008/
_version_	1613495564590120960

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

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