Open and closed structures of L-arginine oxidase by cryo-electron microscopy and X-ray crystallography.

L-arginine oxidase (AROD, EC 1.4.3.

Development of a novel single-chain l-glutamate oxidase from Streptomyces sp. X-119-6 by inserting flexible linkers.

L-glutamate oxidase (LGOX, EC: 1.4.3.

Ambient temperature structure of phosphoketolase from Bifidobacterium longum determined by serial femtosecond X-ray crystallography.

Phosphoketolase and transketolase are thiamine diphosphate-dependent enzymes and play a central role in the primary metabolism of bifidobacteria: the bifid shunt. The enzymes both catalyze phosphorolytic cleavage of xylulose 5-phosphate or fructose 6-phosphate in the first reaction step, but possess different substrate specificity in the second reaction step, where phosphoketolase and transketolase utilize inorganic phosphate (P) and D-ribose 5-phosphate, respectively, as the acceptor substrate. Structures of Bifidobacterium longum phosphoketolase holoenzyme and its complex with a putative inhibitor, phosphoenolpyruvate, were determined at 2.

High-resolution structure of phosphoketolase from Bifidobacterium longum determined by cryo-EM single-particle analysis.

In bifidobacteria, phosphoketolase (PKT) plays a key role in the central hexose fermentation pathway called "bifid shunt." The three-dimensional structure of PKT from Bifidobacterium longum with co-enzyme thiamine diphosphate (ThDpp) was determined at 2.1 Å resolution by cryo-EM single-particle analysis using 196,147 particles to build up the structural model of a PKT octamer related by D symmetry.

Structural insights into the enhanced thermostability of cysteine substitution mutants of L-histidine decarboxylase from Photobacterium phosphoreum.

Enzymatic amino acid assays are important in physiological research and clinical diagnostics because abnormal amino acid concentrations in biofluids are associated with various diseases. L-histidine decarboxylase from Photobacterium phosphoreum (PpHDC) is a pyridoxal 5'-phosphate-dependent enzyme and a candidate for use in an L-histidine quantitation assay. Previous cysteine substitution experiments demonstrated that the PpHDC C57S mutant displayed improved long-term storage stability and thermostability when compared with those of the wild-type enzyme.

Leucine Dehydrogenase: Structure and Thermostability.

Thermostability is a key factor in the industrial and clinical application of enzymes, and understanding mechanisms of thermostability is valuable for molecular biology and enzyme engineering. In this chapter, we focus on the thermostability of leucine dehydrogenase (LDH, EC 1.4.

Quantification of the kokumi peptide, γ-glutamyl-valyl-glycine, in cheese: Comparison between cheese made from cow and ewe milk.

Recent studies have shown that several types of cheese contain kokumi γ-glutamyl dipeptides, and the kokumi tripeptide, γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly), is a component of various fermented foods. The quantification of γ-Glu-Val-Gly in various types of cheese was herein conducted by HPLC-tandem mass spectrometry followed by derivatization with 6-aminoquinoyl-N-hydroxysuccinimidyl-carbamate. The γ-Glu-Val-Gly concentrations were between 0.

Development of a rapid and simple glycine analysis method using a stable glycine oxidase mutant.

Glycine analysis is important in research fields such as physiology and healthcare because the concentration of glycine in human plasma has been reported to change with various disorders. Glycine oxidase from Bacillus subtilis (GlyOX) is useful for quantitative analysis of glycine. However, GlyOX is not sufficiently stable for use in physiology-based research or clinical settings.

Genetic basis for plasma amino acid concentrations based on absolute quantification: a genome-wide association study in the Japanese population.

To assess the use of plasma free amino acids (PFAAs) as biomarkers for metabolic disorders, it is essential to identify genetic factors that influence PFAA concentrations. PFAA concentrations were absolutely quantified by liquid chromatography-mass spectrometry using plasma samples from 1338 Japanese individuals, and genome-wide quantitative trait locus (QTL) analysis was performed for the concentrations of 21 PFAAs. We next conducted a conditional QTL analysis using the concentration of each PFAA adjusted by the other 20 PFAAs as covariates to elucidate genetic determinants that influence PFAA concentrations.

Development of a novel l-histidine assay method using histamine dehydrogenase and a stable mutant of histidine decarboxylase.

l-Histidine analysis is essential in physiological research and clinical applications because l-histidine concentrations in biofluids are associated with various diseases. However, an enzymatic method for l-histidine quantitation has not yet been established. Here, we describe a novel l-histidine quantitation assay using a combination of histidine decarboxylase (HDC) and histamine dehydrogenase (HDH) enzymes.

Structural insights into thermostabilization of leucine dehydrogenase from its atomic structure by cryo-electron microscopy.

Leucine dehydrogenase (LDH, EC 1.4.1.

Protein engineering for improving the thermostability of tryptophan oxidase and insights from structural analysis.

l-Tryptophan oxidase, VioA from Chromobacterium violaceum, which has a high substrate specificity for tryptophan, is useful for quantitative assay of tryptophan. However, stability of wild type VioA is not enough for its application in clinical or industrial use. To improve the thermal stability of the enzyme, we developed a VioA (C395A) mutant, with higher stability than wild type VioA.

Deuteration and selective labeling of alanine methyl groups of β-adrenergic receptor expressed in a baculovirus-insect cell expression system.

G protein-coupled receptors (GPCRs) exist in equilibrium between multiple conformations, and their populations and exchange rates determine their functions. However, analyses of the conformational dynamics of GPCRs in lipid bilayers are still challenging, because methods for observations of NMR signals of large proteins expressed in a baculovirus-insect cell expression system (BVES) are limited. Here, we report a method to incorporate methyl-CH-labeled alanine with > 45% efficiency in highly deuterated proteins expressed in BVES.

Phosphorylation-induced conformation of β-adrenoceptor related to arrestin recruitment revealed by NMR.

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 β-adrenoceptor (βAR) and the phosphorylated βAR-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 M215 and M279, located on transemembrane helices 5 and 6, respectively.

Interaction Analysis of FABP4 Inhibitors by X-ray Crystallography and Fragment Molecular Orbital Analysis.

X-ray crystal structural determination of FABP4 in complex with four inhibitors revealed the complex binding modes, and the resulting observations led to improvement of the inhibitory potency of FABP4 inhibitors. However, the detailed structure-activity relationship (SAR) could not be explained from these structural observations. For a more detailed understanding of the interactions between FABP4 and inhibitors, fragment molecular orbital analyses were performed.

The effects of pre-analysis sample handling on human plasma amino acid concentrations.

Background: The accurate and reliable quantification of amino acid concentrations in human plasma is important for the investigation of a number of diseases. However, few systematic studies investigating the changes in amino acid concentrations related to blood collection and storage conditions have been completed.

Methods: Blood samples were collected with EDTA-Na2 from 3 healthy volunteers and subjected to a number of different treatments; hemolysis, temperature after blood collection, time from blood collection to cooling, the influence of platelets, long term storage conditions, and repeated freeze-thaw cycles.

Validation of an analytical method for human plasma free amino acids by high-performance liquid chromatography ionization mass spectrometry using automated precolumn derivatization.

The analysis of human plasma free amino acids is important for diagnosing the health of individuals, because their concentrations are known to vary with various diseases. The development of valid, reliable, and high-throughput analytical methods for amino acids analysis is an essential requirement in clinical applications. In the present study, we have developed an automated precolumn derivatization amino acid analytical method based on high-performance liquid chromatography/electrospray ionization mass spectrometry (so-called UF-Amino Station).

Simultaneous analysis of D-alanine, D-aspartic acid, and D-serine using chiral high-performance liquid chromatography-tandem mass spectrometry and its application to the rat plasma and tissues.

A highly sensitive and selective chiral LC-MS/MS method for D-alanine, D-aspartic acid and D-serine has been developed using the precolumn derivatization reagents, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ-Tag) or p-N,N,N-trimethylammonioanilyl N'-hydroxysuccinimidyl carbamate iodide (TAHS). The thus N-tagged enantiomers of the derivatized amino acids were nicely separated within 20min using the cinchona alkaloid-based zwittterionic ion-exchange type enantioselective column, Chiralpak ZWIX(+). The selected reaction monitoring was applied for detecting the target d-amino acids in biological matrices.

Determination and quantification of kokumi peptide, γ-glutamyl-valyl-glycine, in brewed alcoholic beverages.

Recently, it has been demonstrated that kokumi substances such as glutathione are perceived through the calcium-sensing receptor (CaSR), and screening by CaSR assay and sensory evaluation has shown that γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly) is a potent kokumi peptide. In this study, γ-Glu-Val-Gly contents in various brewed alcoholic beverages were investigated. Contents of γ-Glu-Val-Gly in four brands of wine, four brands of rice wine (sake) and eight brands of beer were analyzed by high performance liquid chromatography-tandem mass spectrometry followed by derivatization with 6-aminoquinoyl-N-hydroxysuccinimidyl-carbamate.

Microscopic imaging mass spectrometry assisted by on-tissue chemical derivatization for visualizing multiple amino acids in human colon cancer xenografts.

Imaging MS combined with CE/MS serves as a method to provide semi-quantitative and spatial information of small molecular metabolites in tissue slices. However, not all metabolites including amino acids have fully been visualized, because of low-ionization efficiency in MALDI MS. This study aimed to acquire semi-quantitative spatial information for multiple amino acids in frozen tissue slices.

Determination and quantification of the kokumi peptide, γ-glutamyl-valyl-glycine, in commercial soy sauces.

Recent studies have demonstrated that kokumi substances, such as glutathione, are perceived through the calcium-sensing receptor (CaSR), and screening by CaSR assay and sensory evaluation has shown that γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly) is a potent kokumi peptide. In this study, the contents of γ-Glu-Val-Gly in six commercial brands of dark-coloured soy sauces, two brands of light-coloured soy sauce, and one brand of white soy sauce, were investigated by high performance liquid chromatography-tandem mass spectrometry (LC/MS/MS), followed by derivatization with 6-aminoquinoyl-N-hydroxysuccinimidyl-carbamate (AQC). The analyses indicated that γ-Glu-Val-Gly was present in all investigated soy sauces at concentrations ranging from 0.

A new approach for quantitative analysis of L-phenylalanine using a novel semi-sandwich immunometric assay.

Here, we describe a novel method for L-phenylalanine analysis using a sandwich-type immunometric assay approach for use as a new method for amino acid analysis. To overcome difficulties of the preparation of high-affinity and selectivity monoclonal antibodies against L-phenylalanine and the inability to use sandwich-type immunometric assays due to their small molecular weight, three procedures were examined. First, amino groups of L-phenylalanine were modified by "N-Fmoc-L-cysteine" (FC) residues and the derivative (FC-Phe) was used as a hapten.

Determination of γ-glutamyl-valyl-glycine in raw scallop and processed scallop products using high pressure liquid chromatography-tandem mass spectrometry.

The determination of the kokumi peptide, γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly) in raw scallop and processed scallop products was carried out using high pressure liquid chromatography-tandem mass spectrometry (LC/MS/MS). The detection of γ-Glu-Val-Gly was achieved using a multiple reaction monitoring (MRM) method. The optimised condition enabled the precise determination of γ-Glu-Val-Gly.