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

L-glutamate oxidase (LGOX, EC: 1.4.3.11) is an oxidoreductase that catalyzes L-glutamate deamination. LGOX from Streptomyces sp. X-119-6 is used widely for L-glutamate quantification in research and industrial applications. This enzyme encoded as a single precursor chain that undergoes post-translational cleavage to four fragments by an endogenous protease to become highly active. Efficient preparation of active LGOX by heterologous expression without proteolysis process should be indispensable for wide application of this enzyme. Thus, developing an LGOX that requires no protease treatment should expand the potential applications of recombinant LGOX. In this report, we succeeded in obtaining an active single-chain LGOX by connecting the four fragments of the mature form with insertion of flexible linkers. The most active single-chain mutant showed the similar activity to that of the mature form from Streptomyces sp. X-119-6. The structure of this mutant was determined at 2.9 Å resolution by X-ray crystallography. It was revealed that this single-stranded mutant had the similar conformation to that of mature form. This single-chain LGOX can be produced efficiently and should expand LGOX applications.

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 (Pi) 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 2.5 Šresolution by serial femtosecond crystallography using an X-ray free-electron laser. In the complex structure, phosphoenolpyruvate was present at the entrance to the active-site pocket and plugged the channel to thiamine diphosphate. The phosphate-group position of phosphoenolpyruvate coincided well with those of xylulose 5-phosphate and fructose 6-phosphate in the structures of their complexes with transketolase. The most striking structural change was observed in a loop consisting of Gln546-Asp547-His548-Asn549 (the QN-loop) at the entrance to the active-site pocket. Contrary to the conformation of the QN-loop that partially covers the entrance to the active-site pocket (`closed form') in the known crystal structures, including the phosphoketolase holoenzyme and its complexes with reaction intermediates, the QN-loop in the current ambient structures showed a more compact conformation with a widened entrance to the active-site pocket (`open form'). In the phosphoketolase reaction, the `open form' QN-loop may play a role in providing the binding site for xylulose 5-phosphate or fructose 6-phosphate in the first step, and the `closed form' QN-loop may help confer specificity for Pi in the second step.

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 D4 symmetry. Although the cryo-EM structure of PKT was almost identical to the X-ray crystal structure previously determined at 2.2 Å resolution, several interesting structural features were observed in the cryo-EM structure. Because this structure was solved at relatively high resolution, it was observed that several amino acid residues adopt multiple conformations. Among them, Q546-D547-H548-N549 (the QN-loop) demonstrate the largest structural change, which seems to be related to the enzymatic function of PKT. The QN-loop is at the entrance to the substrate binding pocket. The minor conformer of the QN-loop is similar to the conformation of the QN-loop in the crystal structure. The major conformer is located further from ThDpp than the minor conformer. Interestingly, the major conformer in the cryo-EM structure of PKT resembles the corresponding loop structure of substrate-bound Escherichia coli transketolase. That is, the minor and major conformers may correspond to "closed" and "open" states for substrate access, respectively. Moreover, because of the high-resolution analysis, many water molecules were observed in the cryo-EM structure of PKT. Structural features of the water molecules in the cryo-EM structure are discussed and compared with water molecules observed in the crystal structure.

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. In this study, combinational mutation experiments of single cysteine substitution mutants of PpHDC were performed, revealing that the PpHDC C57S/C101V/C282V mutant possessed the highest thermostability. The stabilizing mechanism of these mutations was elucidated by solving the structures of PpHDC C57S and C57S/C101V/C282V mutants by X-ray crystallography. In the crystal structures, two symmetry-related PpHDC molecules form a domain-swapped homodimer. The side chain of S57 is solvent exposed in the structure, indicating that the C57S mutation eliminates chemical oxidation or disulfide bond formation with a free thiol group, thereby providing greater stability. Residues 101 and 282 form hydrophobic interactions with neighboring hydrophobic residues. Mutations C101V and C282V enhanced thermostability of PpHDC by filling a cavity present in the hydrophobic core (C101V) and increasing hydrophobic interactions.

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 1.4.1.9), an amino acid-metabolizing enzyme that is an NAD+-dependent oxidoreductase which catalyzes the deamination of branched-chain l-amino acids (BCAAs). LDH from Geobacillus stearothermophilus (GstLDH) is a highly thermostable enzyme that has already been applied to quantify the concentration of BCAAs in biological specimens. However, the molecular mechanism of its thermostability had been unknown because no high-resolution structure was available. Here, we discuss the thermostability of GstLDH on the basis of its structure determined by cryo-electron microscopy. Sequence comparison with other structurally characterized LDHs (from Lysinibacillus sphaericus and Sporosarcina psychrophila) indicated that non-conserved residues in GstLDH, including Ala94, Tyr127, and the C-terminal region, are crucial for oligomeric stability through intermolecular interactions between protomers. Furthermore, NAD+ binding to GstLDH increased the thermostability of the enzyme as additional intermolecular interactions formed on cofactor binding. This knowledge is important for further applications and development of amino acid metabolizing enzymes in industrial and clinical fields.

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 0.35 and 0.59 µg/g in cheese made from ewe milk, but were not detected in cheese made from cow milk. The amino acid sequences of major milk proteins showed that the ß-caseins of sheep had the Val-Gly sequence at the 9-10 position, whereas ß-caseins of cows contained a Pro-Gly sequence at the same position. The Val-Gly sequence was absent in other caseins of sheep and cattle. These results suggest that the different γ-Glu-Val-Gly concentrations present in cheese made from cow and ewe milk are due to differences in the amino acid sequences of caseins.

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. In this report, site-directed mutagenesis was used to engineer a GlyOX mutant suitable for glycine analysis. The GlyOX triple-mutant (T42 A/C245 S/L301V) retained most of its enzymatic activity during storage for over a year at 4 °C. A colorimetric enzyme analysis protocol was established using the GlyOX triple-mutant to determine glycine concentrations in human plasma. The analysis showed high accuracy (-5.4 to 3.5% relative errors when compared with the results from an amino acid analyzer, and 96.0-98.7% recoveries) and high precision (<4% between-run variation). Sample pretreatments of deproteinization and derivatization were not required. Therefore, this novel enzymatic analysis offers an effective and useful method for determining glycine concentrations in physiology related research and the healthcare field.

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. We identified eight genes that showed a significant association with PFAA concentrations, of which two, SLC7A2 and PKD1L2, were identified. SLC7A2 was associated with the plasma levels of arginine and ornithine, and PKD1L2 with the level of glycine. The significant associations of these two genes were revealed in the conditional QTL analysis, but a significant association between serine and the CPS1 gene disappeared when glycine was used as a covariate. We demonstrated that conditional QTL analysis is useful for determining the metabolic pathways predominantly used for PFAA metabolism. Our findings will help elucidate the physiological roles of genetic components that control the metabolism of amino acids.

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. Wild-type HDC is unstable and completely lost its activity within 50 days of storage at 4 °C in solution. We rationally designed a HDC C57S mutant with markedly improved stability (storage at 4 °C for over 200 days) without altering the enzyme's substrate specificity. Together with HDH, the HDC C57S mutant was applied to quantify l-histidine concentrations in human plasma. The assay showed high precision (<2.0% inter-assay variation) and high accuracy (<5.8% deviation from the results of LC/MS).

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

Leucine dehydrogenase (LDH, EC 1.4.1.9) is a NAD+-dependent oxidoreductase that catalyzes the deamination of branched-chain l-amino acids (BCAAs). LDH of Geobacillus stearothermophilus (GstLDH) is a highly thermostable enzyme that has been applied for the quantification or production of BCAAs. Here the cryo-electron microscopy (cryo-EM) structures of apo and NAD+-bound LDH are reported at 3.0 and 3.2 Šresolution, respectively. On comparing the structures, the two overall structures are almost identical, but it was observed that the partial conformational change was triggered by the interaction between Ser147 and the nicotinamide moiety of NAD+. NAD+ binding also enhanced the strength of oligomerization interfaces formed by the core domains. Such additional interdomain interaction is in good agreement with our experimental results showing that the residual activity of NAD+-bound form was approximately three times higher than that of the apo form after incubation at 80 °C. In addition, sequence comparison of three structurally known LDHs indicated a set of candidates for site-directed mutagenesis to improve thermostability. Subsequent mutation analysis actually revealed that non-conserved residues, including Ala94, Tyr127, and the C-terminal region, are crucial for oligomeric thermostability.

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. The VioA (C395A) exhibited similar specificity and kinetic parameter for tryptophan to wild type. Conventionally, the quantity of tryptophan is determined by instrumental methods, such as high-performance liquid chromatography (HPLC) after pre-column-derivatization. Using the mutant enzyme, we succeeded in the tryptophan quantification in human plasma samples, to an accuracy of <2.9% when compared to the instrumental method, and to a precision of CV <3.2%. To analyse the improvement in storage stability and substrate specificity, we further determined the crystal structures of VioA (C395A) complexed with FAD, and with FAD and tryptophan at 1.8 Å resolution.

Deuteration and selective labeling of alanine methyl groups of ß2-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-13C1H3-labeled alanine with > 45% efficiency in highly deuterated proteins expressed in BVES. Application of the method to the NMR observations of ß2-adrenergic receptor in micelles and in nanodiscs revealed the ligand-induced conformational differences throughout the transmembrane region of the GPCR.

Phosphorylation-induced conformation of ß2-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 ß2-adrenoceptor (ß2AR) and the phosphorylated ß2AR-ß-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 ß-arrestin-bound state. The phosphorylation-induced structures revealed in this study propose a conserved structural motif of GPCRs that enables ß-arrestin to recognize dozens of GPCRs.

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. These analyses revealed that the total interfragment interaction energies of FABP4 and each inhibitor correlated with the ranking of the K i value for the four inhibitors. Furthermore, interactions between each inhibitor and amino acid residues in FABP4 were identified. The oxygen atom of Lys58 in FABP4 was found to be very important for strong interactions with FABP4. These results might provide useful information for the development of novel potent FABP4 inhibitors.

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. Changes in the concentrations of 22 amino acids were determined using an Amino Acid Analyzer. RESULTS: Of the conditions influencing sample stability between blood collection and amino acid analysis, hemolysis, temperature after blood collection, and long-term storage at -20°C affected the concentrations of 11 amino acids. Time from blood collection to cooling, platelet contamination and repeated freeze-thaw cycles altered the levels of 4 amino acids. CONCLUSIONS: We observed changes in amino acid concentrations relating to blood collection and storage conditions. If attention is paid to 4 key factors (hemolysis, temperature immediately following blood collection, time from collection to cooling, and long-term storage temperature) 19 amino acids can be reliably quantified.

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. By using the present chiral LC-MS/MS method, the three d-amino acids and their l-forms could be simultaneously determined in the range of 0.1-500nmol/mL. Finally, the technique was successfully applied to rat plasma and tissue samples.

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). This method enabled the separation of at least 38 types of physiological amino acids within 8min, and the interval time between injections was 12min. We also validated this method for 21 major types of free amino acids in human plasma samples. The results of the specificity, linearity, accuracy, repeatability, intermediate precision, reproducibility, limits of detections, lower limits of quantification, carry over, and sample solution stability were sufficient to allow for the measurement of amino acids in human plasma samples. Our developed method should be suitable for use in clinical fields.

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. The analyses indicated that γ-Glu-Val-Gly was present in all of eight beer samples at concentrations in the range of 0.08-0.18 mg/L, although the peptide was not detected in any wine or rice wine samples. These results suggest that amongst the brewed beverages tested, beer contains γ-Glu-Val-Gly, and that γ-Glu-Val-Gly is widely distributed in beer.