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, Kunio; Ohshima, Toshihisa

doi:10.1186/s40064-015-1335-6

Cited by 6 publications

(12 citation statements)

References 30 publications

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“…Results indicate that the rALRase is stable at 4–40°C. In addition, L. salivarius with ALRase had heat tolerance for 30 min at 50°C: 70% of its activity was retained, as reported by Kobayashi et al (2015). Furthermore, ALRase having Bacillus anthracis had heat tolerance, even at 60°C, at which 60% of its activity remained (Kanodia et al, 2009).…”

Section: Resultssupporting

confidence: 63%

“…and Geobacillus spp. : 129th lysine in amino acid residue (K129), 138th arginine in amino acid residue (R138), 314th methionine in amino acid residue (M314), and 315th aspartic acid in amino acid residue (D315) (Kanodia et al, 2009; Kobayashi et al, 2015; Morollo et al, 1999). Kobayashi et al (2015) reported histidine and leucine in approximately the 130th amino acid residue as H129 and L130 in ALRmase in Lactobacillus salivarius , and H127 and L128 in that of the Bacillus spp.…”

Section: Resultsmentioning

confidence: 99%

“…For instance, for ALRase in L. sakei NBRC15893 isolated from Kimoto as sake seed mash, Kato and Oikawa (2018) reported optimum conditions of pH 10.0 and 45°C. Moreover, Kobayashi et al (2015) reported optimum pH for L. salivarius with ALRase as 8.0. The optimum conditions of ZH‐2 agree with those for L. sakei NBRC15893.…”

Section: Resultsmentioning

confidence: 99%

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Characteristics of alanine racemase in Lactobacillus sakeiZH‐2 strain

Kanauchi

Matsumoto

2023

Food Science & Nutrition

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Some d‐amino acid functions for food production are widely known: d‐alanine improves sensory evaluations of sake, beer, and fermented foods. Therefore, for the application of d‐amino acids, alanine racemase (ALRase) in Lactobacillus sakei ZH‐2, which has strong racemization, was analyzed using molecular biological methods. It had been hypothesized that ALRase coding DNA, alr, in ZH‐2 strain differs from those of other Lactobacillus sakei strains. However, complete genome sequencing by the National Center for Biotechnology (NCBI) revealed the amino acid sequence of alr in ZH‐2 strain to have homology of 99.4% similarity with the alr in Lactobacillus sakei 23K strain. However, it is considered that the sequence of alr was a unique amino acid sequence in the lactic acid bacteria group. DNA “alr” of ZH‐2 strain has a 1140 bp DNA base with 41 kDa molecular mass. Its molecular mass was inferred as approximately 38.0 kDa using SDS‐PAGE. Its optimum conditions are pH 9.0 at 30–40°C, showing stability at pH 9.0–10.0 and 4–40°C. Its cofactor is pyridoxal phosphate. Its activity is activated more by copper and zinc ions than by the lack of a metal ion. Additionally, its Km is 1.32 × 10−3 (mol), with Vmax of 4.27 × 10−5 (μmol−1 min−1). ALRase reacted against alanine most strongly in other substrates such as amino acids. The enzyme against serine was found to have 40% activity against alanine. The enzyme converted up to 54.5% of d‐alanine from l‐alanine ZH‐2 strain.

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Section: Resultssupporting

confidence: 63%

Section: Resultsmentioning

confidence: 99%

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Characteristics of alanine racemase in Lactobacillus sakeiZH‐2 strain

Kanauchi

Matsumoto

2023

Food Science & Nutrition

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“…Thus, the relationship between d-amino acids and lactic acid bacteria should be further studied. Research into the enzymatic properties of these racemases has revealed a unique characteristic of AlaR from Lactobacillus salivarius UCC118 (LsAlaR) [49]. The presence of short-chain carboxylates, such as acetate and propionate, stabilizes and activates the LsAlaR, and these carboxylates generally inhibit the racemization activity of AlaR as substrate analogs [50].…”

Section: D-amino-acid-metabolizing Enzymes In Lactic Acid Bacteriamentioning

confidence: 99%

d-Amino Acids and Lactic Acid Bacteria

Kobayashi

2019

Microorganisms

Self Cite

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Proteins are composed of l-amino acids except for glycine, which bears no asymmetric carbon atom. Accordingly, researchers have studied the function and metabolism of l-amino acids in living organisms but have paid less attention to the presence and roles of their d-enantiomers. However, with the recent developments in analytical techniques, the presence of various d-amino acids in the cells of various organisms and the importance of their roles have been revealed. For example, d-serine (d-Ser) and d-aspartate (d-Asp) act as neurotransmitters and hormone-like substances, respectively, in humans, whereas some kinds of d-amino acids act as a biofilm disassembly factor in bacteria. Interestingly, lactic acid bacteria produce various kinds of d-amino acids during fermentation, and many d-amino acids taste sweet, compared with the corresponding l-enantiomers. The influence of d-amino acids on human health and beauty has been reported in recent years. These facts suggest that the d-amino acids produced by lactic acid bacteria are important in terms of the taste and function of lactic-acid-fermented foods. Against this background, unique d-amino-acid-metabolizing enzymes have been searched for and observed in lactic acid bacteria. This review summarizes and introduces the importance of various d-amino acids in this regard.Proteins are basically made up of 20 kinds of α-amino acids in the form of monomers, and these amino acids have at least one asymmetric carbon, except for glycine. This yields 19 kinds of amino acid (glycine has been excepted) with l-and d-form varieties. As proteins are made up of l-amino acids, many researchers, in fields such as medical, food, and nutritional science, have paid close attention to l-amino acids, giving rise to considerable knowledge of l-amino acids, including proteins. Until the 1970s, many researchers thought that d-amino acids were rare in nature and did not play an important role in living organisms. After that, however, d-alanine (d-Ala), d-glutamate (d-Glu), and d-aspartate (d-Asp) were found in bacterial cell walls as constituents, and d-amino acids were observed to exist in nature. This finding led to the conclusion that d-amino acids were only rarely present in bacterial cell walls, but in the 1990s, the development of analytical techniques, such as chiral column chromatography, meant that various kinds of d-amino acids were identified in free forms in diverse organisms. These d-amino acids were initially considered to be derived from foods or enterobacteria, but it is now known that some parts of d-amino acids such as d-serine (d-Ser) and d-Asp can be synthesized in mammalian cells. d-Amino acids have similar physical and chemical characteristics with l-amino acids, but the physiological functions of d-amino acids are completely different with those of l-amino acids. For example, d-Ser works as neurotransmitter in the brain, but l-Ser has no such role. Furthermore, d-amino acids are uncommon in living organisms, compared with l-amino acids, and despite the fact that some d-...

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“…A lysine residue at position 129 in ALR from Geobacillus stearothermophilus is conserved in many well-studied ALRs, whereas it is replaced with alanine in ALR from Ligilactobacillus salivarius. Previous studies indicate that short-chain carboxylates inhibit ALRs from G. stearothermophilus and Bacillus anthracis by acting as substrate analogues, whereas L. salivarius ALR is activated by carboxylates, such as acetate and propionate [10][11][12]. Replacement of this alanine with lysine at position 129 in L. salivarius ALR prevents the enzyme from being activated by the carboxylates, indicating that this residue plays a crucial role in activation/inhibition, although the associated mechanism remains unclear.…”

Section: Introductionmentioning

confidence: 99%

Regulation of alanine racemase activity by carboxylates and the d‐type substrate d‐alanine

et al. 2023

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Alanine racemases (ALRs) are essential for D-alanine (D-Ala) production in bacteria, and many ALRs have a conserved carbamylated lysine residue in the active site. Although short-chain carboxylates inhibit ALRs harbouring this lysine residue as substrate analogues, in an ALR variant with an alanine residue at this position, carboxylates behave as activators; however, this activation mechanism remains unclear. Here, we performed kinetic and structural analyses of U1ALR, an ALR from Latilactobacillus sakei UONUMA harbouring a glycine residue (Gly134) in the site of the carbamylated lysine residue. U1ALR was activated by various carboxylates and also by a G134K mutation, both of which caused a significant decrease in K m , indicating an increase in substrate affinity. The U1ALR crystal structure revealed the presence of an acetate molecule bound in a position and at an orientation resembling the conformation of the carbamylated lysine side chain observed in the structures of other ALRs. These results suggest a regulatory mechanism for U1ALR activity involving two carboxylatebinding sites: one with high affinity near Gly134, where an acetate molecule is observed in the crystal structure and carboxylate binding results in enzyme activation; the other is the substrate-binding site, where carboxylate binding inhibits enzyme activity. Furthermore, we observed no carboxylate/G134K-mediated activation in the presence of D-Ala at high concentrations, implying that D-Ala also exhibits low-affinity binding in the first carboxylate-binding site and prevents carboxylate/G134K-induced activation. Such regulation of enzyme activity by carboxylates and D-Ala may be ubiquitous in many ALRs from lactic acid bacteria sharing the same sequence characteristics.

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

Cited by 6 publications

References 30 publications

Characteristics of alanine racemase in Lactobacillus sakeiZH‐2 strain

Characteristics of alanine racemase in Lactobacillus sakeiZH‐2 strain

d-Amino Acids and Lactic Acid Bacteria

Regulation of alanine racemase activity by carboxylates and the d‐type substrate d‐alanine

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