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L-Phenylalanine (L-Phe), which is one of the essential amino acids, is important for the human body because it is used in the biosynthesis of neurotransmitters. Phenylalanine dehydrogenase (PheDH) has been studied as a material for the L-Phe sensor. In particular, the accurate and simple determination of L-Phe concentrations in human samples is needed for the diagnosis and treatment of patients with phenylketonuria. Thermostable PheDH from the bacterium Thermoactinomyces intermedius is suitable as a sensor material; however, it is reactive to not only L-Phe but also L-tyrosine (L-Tyr). Thus, we attempted to improve its substrate specificity for the accurate measurement of L-Phe. In this study, we developed the PheDH mutant with improved substrate specificity by amino acid substitution. The PheDH mutant, in which the 290th asparagine residue was substituted with aspartic acid (N290D), is considered to form a new interaction in the substrate binding site and has a markedly reduced reactivity with L-Tyr compared with the wild-type PheDH; the relative activities (L-Tyr/L-Phe) of the wild-type PheDH and the PheDH N290D mutant were 64.0 and 1.5%, respectively. An enzymatic assay using this mutant was established to quantify the L-Phe concentration in human plasma. The relative errors ranged from −10.3 to 7.4% when compared with the quantitative value determined by high-performance liquid chromatography/electrospray ionization mass spectrometry. Furthermore, the immobilized mutant showed a concentration-dependent reactivity with L-Phe and no reactivity with L-Tyr, which indicate its potential use as a sensor material. [ABSTRACT FROM AUTHOR]

l-glutamate is an umami component found in various foods, and l-glutamate oxidase (LGOX, EC 1.4.3.11) is a FAD-dependent oxidoreductase that catalyzes the oxidative deamination of l-glutamate. A recently reported single-chain LGOX mutant from Streptomyces sp. X-119-6 is a potential material for l-glutamate sensors because this mutant is easier to prepare than the wild type. Herein, we report the development of a simple analytical kit using this LGOX mutant for the on-site quantification of l-glutamate. The kit named PACKTEST was developed using a colorimetric method to estimate the l-glutamate concentration in the range of 1–50 mg/L through the visual comparison of the resulting sample color with a standard color sequence. In addition, the PACKTEST kit was combined with a portable photometer for the spectrophotometric quantification of l-glutamate in the range of 0.5–12.0 mg/L. It was used to quantify l-glutamate in commercially available soy sauces, and the results were in good agreement (r = 0.97) with the values obtained by post-column derivatized ion-exchange chromatography. Furthermore, we loaded and dried all the reagents, including enzymes, on a 96- well plate to prepare an enzyme-based sensing device, which showed potential for the high-throughput quantification of l-glutamate. [ABSTRACT FROM AUTHOR]

Upon stimulation by agonist binding, the C-terminal regions of G-protein-coupled receptors (GPCRs) become phosphorylated by GPCR kinases, and phosphorylated GPCRs bind arrestin. Here the authors give structural insights into the phosphorylation induced conformational changes in GPCRs by performing NMR studies with the β2-adrenoceptor.

The C-terminal region of G-protein-coupled receptors (GPCRs), stimulated by agonist binding, is phosphorylated by GPCR kinases, and the phosphorylated GPCRs bind to arrestin, leading to the cellular responses. To understand the mechanism underlying the formation of the phosphorylated GPCR-arrestin complex, we performed NMR analyses of the phosphorylated β2-adrenoceptor (β2AR) and the phosphorylated β2AR–β-arrestin 1 complex, in the lipid bilayers of nanodisc. Here we show that the phosphorylated C-terminal region adheres to either the intracellular side of the transmembrane region or lipids, and that the phosphorylation of the C-terminal region allosterically alters the conformation around M2155.54 and M2796.41, located on transemembrane helices 5 and 6, respectively. In addition, we found that the conformation induced by the phosphorylation is similar to that corresponding to the β-arrestin-bound state. The phosphorylation-induced structures revealed in this study propose a conserved structural motif of GPCRs that enables β-arrestin to recognize dozens of GPCRs. [ABSTRACT FROM AUTHOR]

The content of a kokumi peptide, γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly), in several kinds of commercial fermented shrimp paste condiments was determined using a LC/MS/MS method after derivatization with 6-aminoquinoyl-N-hydroxy-succinimidyl-carbamate (AQC). Commercial fermented shrimp paste condiments from Indonesia (Terasi), the Philippines (Bagoong) and China (Xiajiang) were analyzed. The analyses revealed that the concentration of γ-Glu-Val-Gly in Terasi, Bagoong, and Xiajiang was 5.2 µg/g, 1.0 µg/g, and 0.9 µg/g, respectively. These results suggested that µ-Glu-Val-Gly is widely distributed in fermented shrimp paste condiments. [ABSTRACT FROM AUTHOR]