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

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 Km , 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 carboxylate-binding 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.

New Inhibitory Effect of Latilactobacillus sakei UONUMA on the Cholesterol Biosynthesis Pathway in Human HepG2 Cells.

Many pharmaceuticals and dietary foods have been reported to inhibit cholesterol biosynthesis, mainly by inhibiting the presqualene enzyme 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase rather than a postsqualene enzyme. In this study, we examined the inhibitory effects of Latilactobacillus sakei UONUMA on cholesterol biosynthesis, especially postsqualene, in human HepG2 hepatoma cells. We quantified cholesterol and its precursors, and the mRNA and protein levels of enzymes involved in cholesterol biosynthesis. Three L. sakei UONUMA strains exhibited new inhibitory effects on cholesterol biosynthesis and inhibited the mRNA level of sterol-delta24-reductase (DHCR24), which is involved in the postsqualene cholesterol biosynthesis pathway. These strains will be useful for the prevention and treatment of hyperlipidemia.

Metabolite profile of koji amazake and its lactic acid fermentation product by Lactobacillus sakei UONUMA.

The koji amazake is a traditional sweet Japanese beverage. It has been consumed for over a thousand years in Japan; nonetheless, little is yet known of the ingredients in koji amazake. Therefore, this study aimed to analyze the metabolites of koji amazake using a metabolomics approach. Additionally, we reformed the flavor of koji amazake by lactic acid fermentation (LAF-amazake) using Lactobacillus sakei UONUMA, which was isolated from snow caverns. The purpose of this article is to identify the ingredients in these beverages. In LAF-amazake and koji amazake, sugars, amino acids, organic acids, and vitamin B complex were determined in the two beverages, and over 300 compounds were detected in total. Thirteen saccharides were identified including two unknown trisaccharides, and there were no differences in these between the two beverages. In LAF-amazake, lactic acid, vitamin B2 (riboflavin), B3 (nicotinic acid and nicotinamide), and B6 (pyridoxine) were significantly increased as compared to koji amazake, whereas malate and glutamine decreased. These results suggested that LAF, malolactic fermentation, and glutamine deamidation occurred simultaneously in LAF-amazake. L. sakei UONUMA strains produced these vitamins. Moreover, it was surprising that acetylcholine, a well-known neurotransmitter, was newly generated in LAF-amazake. Here, we have succeeded in reforming the flavor of koji amazake and obtained these metabolic data on the two beverages. The present study could provide useful basic information for promoting functional analyses of koji amazake and LAF-amazake for human health.

Properties and substrate specificities of proteolytic enzymes from the edible basidiomycete Grifola frondosa.

Highly active proteolytic enzymes are found in the fruiting bodies of Grifola frondosa. The general properties and substrate specificities of these proteases from G. frondosa (ProGF) were studied. The optimal pH for ProGF activity was pH 3 or 7 using hemoglobin or Hammersten casein as a substrate, respectively. The ProGF exhibited over 70% of maximal activity within the pH range of 4.5-8.5. In terms of temperature, the ProGF were maximally active at 55 degrees C, while over 80% of maximal activity was observed within the range of 50-75 degrees C. These proteases were substrate-specific, mainly cleaving at Ala(14)-Leu(15), Tyr(16)-Leu(17), and Pro(28)-Lys(29) bonds, with occasional cleavage of Phe(24)-Phe(25) bonds in the oxidized insulin B-chain. The ProGF also liberated hydrophobic amino acids, such as valine, leucine, and phenylalanine, using the oxidized insulin B-chain as a substrate. When soy protein was used as a substrate, valine, leucine, phenylalanine, and tyrosine were selectively released from the hydrolysate. Thus, over the time course of incubation, the peptide concentration increased as the average peptide chain length decreased. These results indicate that the ProGF include both endopeptidases recognizing leucine, phenylalanine, and lysine at the P1' position, and aminopeptidases preferentially releasing hydrophobic and aromatic amino acids such as valine, leucine, phenylalanine, and tyrosine.

Contamination of the Shinano River water with mutagenic substances after the Niigata Chuetsu Earthquake.

While normally monitoring the Shinano River water quality, including examinations for mutagenicity, the Niigata Chuetsu Earthquake suddenly occurred on October 23, 2004. However, the influence of this earthquake on the mutagenicity of river water has not yet been well studied. To clarify the regional and seasonal changes in mutagenicity of the Shinano River water, blue rayon was suspended for 24 hrs at 4 sampling sites, once a month from September 2004 through August 2005. Mutagenicity was evaluated by the Ames test using Salmonella typhimurium TA98 (TA98) and TA100 with or without metabolic activation by S9 mixture. To detect and identify poly-aromatic hydrocarbons that may be responsible for the mutagenicity of the river water, we analyzed benzo[a]pyrene, benzophenone, 4-nitrotoluene, or other compounds using gas chromatography-mass spectrometry and total ion chromatogram spectra. Positive manifestations of TA98 with S9 mixture were observed at the 4 sampling sites throughout the 12-month test, showing a tendency to be higher at the downstream site and in winter. However, the highest mutagenicity was observed in the sample collected at the most upstream sampling site in December 2004, and fluoranthene or pyrene consisting mainly in coal tar was detected only in the samples collected in December 2004. Although benzo[a]pyrene, benzophenone, and 4-nitrotoluene were below the detection limits, non-mutagens such as aliphatic hydrocarbons or esters were frequently detected. Our findings indicate that either fluoranthene or pyrene was mainly responsible for the mutagenicity of the river water in December 2004, suggesting the possibility of oil contamination caused by the Niigata Chuetsu Earthquake.

Debittering of enzymatic hydrolysates using an aminopeptidase from the edible basidiomycete Grifola frondosa.

Bitter peptide solutions, prepared by the enzymatic hydrolysis of soy protein and milk casein, were treated with an aminopeptidase from the edible basidiomycete Grifola frondosa. As the incubation time elapsed, the amount of free amino acids released increased and the bitterness of the enzyme reaction mixtures decreased. However, the debittering of the milk casein hydrolysate by the aminopeptidase was less effective than that observed for the soy protein hydrolysate. Hydrophobic amino acids such as valine, leucine, phenylalanine, tyrosine, and isoleucine were preferentially released from the bitter solutions by the action of the aminopeptidase.