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.

Analytical Chemistry of Impurities in Amino Acids Used as Nutrients: Recommendations for Regulatory Risk Management.

Proteinogenic amino acids are natural nutrients ingested daily from standard foods. Commercially manufactured amino acids are added to a wide range of nutritional products, including dietary supplements and regular foods. Currently, the regulatory risk management of amino acids is conducted by means of setting daily maximum limits of intake. However, there have been no reported adverse effects of amino acid overdosing, while impurities in low-quality amino acids have been identified as causative agents in several health hazard events. This paper reviews the analytical chemistry of impurities in amino acids and highlights major variations in the purity of commercial products. Furthermore, it examines the international standards and global regulatory risk assessment of amino acids utilized in dietary supplements and foods, recommending (1) further research on analytical methods that can comprehensively separate impurities in amino acids, and (2) re-focusing on the regulatory risk management of amino acids to the analytical chemistry of impurities.

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.

Lung cancer screening by single-shot dual-energy subtraction using flat-panel detector.

PURPOSE: The purpose of this study was to evaluate the usefulness of single-shot dual-energy subtraction (DES) method using a flat-panel detector for lung cancer screening MATERIALS AND METHODS: The subjects were 13,315 residents (5801 males and 7514 females) aged 50 years or older (50-97 years, with an intermediate value of 68 years) who underwent lung cancer screening for a period of 1 year and 6 months from January 2019 to June 2020. We investigated whether the number of lung cancers detected, the detection rate, and the rate of required scrutiny changed, when DES images were added to the judgment based on conventional chest radiography. RESULTS: When DES images were added, the number and percentage of cancer detection increased from 16 (0.12%) to 23 (0.17%) (P < 0.05). Five of the newly detected 7 lung cancers were in the early stages of resectable cancer. The rate of participants requiring scrutiny increased slightly from 1.1 to 1.3%. CONCLUSION: DES method improved the detection of lung cancer in screening. The increase in the percentage of participants requiring scrutiny was negligible.

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.

Biaryl axially chiral derivatizing agent for simultaneous separation and sensitive detection of proteinogenic amino acid enantiomers using liquid chromatography-tandem mass spectrometry.

Novel sophisticated derivatizing agents for the efficient enantioselective separation and mass spectrometric detection of d- and l-amino acids have been developed. Two new axially chiral reagents derived from 6,6'-dimethyl-2,2'-biphenyldiamine were synthesized. Their chiral separation and detection abilities were evaluated by derivatizing proteinogenic amino acid standards in reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS). The resulting diastereomers derived from the developed derivatizing agents and amino acids could be completely separated, because of the effective chiral environment constructed by the axially chiral biphenyl moiety. After optimizing the reactive group, (R)-4-nitrophenyl N-[2'-(diethylamino)-6,6'-dimethyl-[1,1'-biphenyl]-2-yl]carbamate hydrochloride ((R)-BiAC) was found to be the best reagent for highly sensitive simultaneous d,l-amino acid analysis. Using (R)-BiAC, the complete chiral separation of all derivatized proteinogenic amino acids was achieved within 11.5 min with Rs greater than 1.9, except for certain allo-isomers. An exceptional feature of this reagent was its control of elution order, i.e., it afforded elution of the diastereomers derived from d-amino acids before their l-amino acid counterparts for all 19 proteinogenic amino acids. Sensitive detection was also achieved by introducing a dialkyl amino group and selectively cleaving it at the binding site between the reagent and amino acid. Attomole (amol) detection limits (signal-to-noise ratio = 3) were obtained for the tested d,l-amino acids, in the range 7.0-127 amol. As an example of application, the method was applied to food sample analysis, and detected several d-amino acids. Consequently, the developed method seems likely to facilitate simultaneous determination of enantiomers, including the tiny amounts of d-amino acids found in nature.

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).

Plasma amino acid profile associated with fatty liver disease and co-occurrence of metabolic risk factors.

Fatty liver disease (FLD) increases the risk of diabetes, cardiovascular disease, and steatohepatitis, which leads to fibrosis, cirrhosis, and hepatocellular carcinoma. Thus, the early detection of FLD is necessary. We aimed to find a quantitative and feasible model for discriminating the FLD, based on plasma free amino acid (PFAA) profiles. We constructed models of the relationship between PFAA levels in 2,000 generally healthy Japanese subjects and the diagnosis of FLD by abdominal ultrasound scan by multiple logistic regression analysis with variable selection. The performance of these models for FLD discrimination was validated using an independent data set of 2,160 subjects. The generated PFAA-based model was able to identify FLD patients. The area under the receiver operating characteristic curve for the model was 0.83, which was higher than those of other existing liver function-associated markers ranging from 0.53 to 0.80. The value of the linear discriminant in the model yielded the adjusted odds ratio (with 95% confidence intervals) for a 1 standard deviation increase of 2.63 (2.14-3.25) in the multiple logistic regression analysis with known liver function-associated covariates. Interestingly, the linear discriminant values were significantly associated with the progression of FLD, and patients with nonalcoholic steatohepatitis also exhibited higher values.

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.

Validity of a Self-Administered Food-Frequency Questionnaire for Assessing Amino Acid Intake in Japan: Comparison With Intake From 4-Day Weighed Dietary Records and Plasma Levels.

BACKGROUND: Interest in the physiological roles of amino acids and their impact on health outcomes is substantial and growing. This interest has prompted assessment of the habitual intake of amino acids for use in epidemiologic studies and in clarifying the association between habitual intake and plasma levels of amino acids. Here, we investigated the validity of ranking individuals according to dietary amino acid intake as estimated using a food frequency questionnaire (FFQ) in comparison with intakes from dietary records (DRs) and plasma levels. METHODS: A total of 139 men and women selected from examinees of the cancer screening program at the Research Center for Cancer Prevention and Screening, National Cancer Center, Japan, provided 4-day weighed DRs, a semi-quantitative FFQ, and plasma samples. Plasma levels of amino acids were measured using the UF-Amino Station system. RESULTS: Spearman rank correlation coefficients of energy-adjusted intake of amino acids from the DR and FFQ ranged from 0.40 to 0.65 for men and from 0.35 to 0.46 for women. Correlation coefficients of energy-adjusted intake from the DR and plasma levels ranged from -0.40 to 0.25 for men and from -0.16 to 0.11 for women. Similarly, no significant positive correlation coefficients were observed between intake from the FFQ and plasma levels for either men or women. CONCLUSIONS: We confirmed that this FFQ has moderate validity in estimating amino acid intake when 4-day weighed DRs are used as a reference method, suggesting that it is suitable for ranking individuals living in urban areas in Japan by amino acid intake.

Plasma amino acid profiles in healthy East Asian subpopulations living in Japan.

OBJECTIVES: Profiles of plasma free amino acids (PFAAs) have been utilized as biomarkers to detect various diseases. However, few studies have investigated whether ethnicity or specific subpopulations within East Asia influence PFAA concentrations. METHODS: A total of 95 healthy volunteers living in Japan, including 31 Japanese individuals, 36 Korean individuals and 28 Chinese individuals, were enrolled. Participants' PFAA levels were measured by high-performance liquid chromatography mass spectrometry, and the effects of factors such as sex, age, body mass index (BMI) and subpopulation on PFAA profiles were analyzed. RESULTS: With the exception of glutamine and α-aminobutyric acid, there were no significant differences among the three examined subpopulations with respect to either the means or the distributions of PFAA concentrations. A multiple regression analysis revealed that most of the PFAA concentrations were significantly related to sex. Ornithine concentrations, glutamate concentrations, and glutamine and α-aminobutyric acid concentrations were significantly associated with age, BMI, and Chinese subpopulation, respectively. CONCLUSION: The study results indicate that the contributions of subpopulation within East Asia to PFAA profiles are small, particularly relative to the contributions provided by sex.

Reference intervals for plasma-free amino acid in a Japanese population.

BACKGROUND: Plasma amino acid concentrations vary with various diseases. Although reference intervals are useful in daily clinical practice, no reference intervals have been reported for plasma amino acids in a large Japanese population. METHODS: Reference individuals were selected from 7685 subjects examined with the Japanese Ningen Dock in 2008. A total of 1890 individuals were selected based on exclusion criteria, and the reference samples were selected after the outlier samples for each amino acid concentration were excluded. The lower limit of the reference intervals for the plasma amino acid concentrations was set at the 2.5th percentile and the upper limit at the 97.5th percentile. RESULTS: By use of the nested analysis of variance, we analysed a large dataset of plasma samples and the effects of background factors (sex, age and body mass index [BMI]) on the plasma amino acid concentrations. Most amino acid concentrations were related to sex, especially those of branched-chained amino acid. The citrulline, glutamine, ornithine and lysine concentrations were related to age. The glutamate concentration was related to body mass index. CONCLUSIONS: The concentrations of most amino acids are more strongly related to sex than to age or body mass index. Our results indicate that the reference intervals for plasma amino acid concentrations should be stratified by sex when the background factors of age and body mass index are considered.

The performance of a novel amino acid multivariate index for detecting lung cancer: A case control study in Korea.

INTRODUCTION: Previous studies have shown that plasma free amino acid (PFAA) profiles are altered in cancer patients compared with healthy controls. A multivariate index based on PFAAs was generated from a Japanese dataset and has been previously demonstrated to be clinically valuable for discriminating patients in the early stages of lung cancer. However, it remains unclear whether similar PFAA profile changes occur in cancer patients from other populations. Therefore, this study aimed to validate the performance of this index in discriminating lung cancer patients from controls in the Korean population. METHODS: Samples were collected from a total of 142 Korean subjects (72 lung cancer/70 controls) for this study. PFAAs were quantified by high-performance liquid chromatography-electrospray ionization-mass spectrometry, and the clinical performance characteristics of the amino acid multivariate index were evaluated across cancer stages and histological types. RESULTS: The concentrations of several PFAAs were significantly decreased in the Korean lung cancer patients compared with the controls. Significant decreases in threonine, citrulline, histidine and tryptophan and increases in proline, isoleucine, phenylalanine and ornithine were observed, which are similar to the PFAA changes reported by a previous Japanese study. The area under the receiver-operator characteristic curve (AUC of the ROC) for the index was 0.80, and similar performances were demonstrated for the different histological types. CONCLUSIONS: These results suggest that the amino acid multivariate index previously developed from a Japanese dataset has the potential to aid in the early detection of lung cancers of different histological types in Korean patients.