A Glycopeptidyl-Glutamate Epimerase for Bacterial Peptidoglycan Biosynthesis.

d-Glutamate (Glu) supplied by Glu racemases or d-amino acid transaminase is utilized for peptidoglycan biosynthesis in microorganisms. Comparative genomics has shown that some microorganisms, including Xanthomonas oryzae, perhaps have no orthologues of these genes. We performed shotgun cloning experiments with a d-Glu auxotrophic Escherichia coli mutant as the host and X. oryzae as the DNA donor. We obtained complementary genes, XOO_1319 and XOO_1320, which are annotated as a hypothetical protein and MurD (UDP-MurNAc-l-Ala-d-Glu synthetase), respectively. By detailed in vitro analysis, we revealed that XOO_1320 is an enzyme to ligate l-Glu to UDP-MurNAc-l-Ala, providing the first example of MurD utilizing l-Glu, and that XOO_1319 is a novel enzyme catalyzing epimerization of the terminal l-Glu of the product in the presence of ATP and Mg2+. We investigated the occurrence of XOO_1319 orthologues and found that it exists in some categories of microorganisms, including pathogenic ones.

Phenolic compounds responsible for the superoxide dismutase-like activity in high-Brix apple vinegar.

High-Brix apple vinegar (HBAV) with palatable drinking qualities has been developed using a greater amount of apple ingredients. In HBAV and in regular apple vinegar (RAV), constituents of 4 kinds of organic acids, 20 kinds of amino acids, 3 kinds of sugars, 4 kinds of minerals, and phenols were determined. These constituents, except for acetic acid, in HBAV are of higher abundance than in RAV. HBAV had a 7.1 times greater superoxide dismutase (SOD)-like activity compared with RAV. Those constituents, except for phenols, had very low SOD-like activity, and total phenol levels in HBAV were comparable to 181 mg of gallic acid equivalents/100 mL, which was 6.0 times more abundant than in RAV. Nine kinds of phenols including two kinds of hydroxycinnamates, two kinds of hydroxybenzoates, and five kinds of hydroxycinnamoyl quinates, originating from raw material were determined, but there were no ascorbic acid and flavonoids in HBAV. Chlorogenic acid, 4-p-coumaroylquinic acid, and caffeic acid were the three major phenols, and their content levels were 19.6, 13.5, and 0.76 mg in 100 mL of HBAV, respectively. Sum of contents of chlorogenic acid and the isomers was 24.0 mg/100 mL, and the percentage was 56.9% in the total identified phenols in HBAV. In RAV, only chlorogenic acid was determined as phenols, and the content was 3.1 mg/100 mL. SOD-like activities of the constituents of HBAV were obtained through high-accuracy assays using vinegar reconstitutions, and each contribution to the total SOD-like activity was found. As a result, 77.2% for all SOD-like activity of HBAV was reconstituted using the determined nine phenols and other constituents. Chlorogenic acids were the most effective, and the contribution to the total activity was 41.7%. The most abundant phenols, chlorogenic acids, were the most important contributors to the SOD-like activity. These SOD-active phenols originated from raw material and remained through the acetic acid fermentation processes. In the fermentation process of HBAV, the active constituents were well maintained, providing an advantage in the production of a phenol-rich product.