Enzymes in riboflavin biosynthesis: Potential antibiotic drug targets.

The rapid resistance of pathogens to antibiotics has emerged as a major threat to global health. Identification of new antibiotic targets is thus needed for developing alternative drugs. Genes encoding enzymes involved in the biosynthesis of riboflavin and flavin cofactors (FMN/FAD) are attractive targets because these enzymatic reactions are necessary for most bacteria to synthesize flavin cofactors for use in their central metabolic reactions. Moreover, humans lack most of these enzymes because we uptake riboflavin from our diet. This review discusses the current knowledge of enzymes involved in bacterial biosynthesis of riboflavin and other flavin cofactors, as well as the functions of the FMN riboswitch. Here, we highlight recent progress in the structural and mechanistic characterization, and inhibition of GTP cyclohydrolase II (GCH II), lumazine synthase (LS), riboflavin synthase (RFS), FAD synthetase (FADS), and FMN riboswitch, which have been identified as plausible antibiotic targets. As the structures and functions of these enzymes and regulatory systems are not completely understood, they are attractive as subjects for future in-depth biochemical and biophysical analysis.

Effective formative assessment for pharmacy students in Thailand: lesson learns from a school of pharmacy in Thailand.

INTRODUCTION: Formative assessment (FA) is an assessment concept that is of interest in education. The Doctor of Pharmacy program is one of the programs in which FA is usually implemented. This study aimed to describe the correlation between FA scores and summative assessment (SA) scores and to suggest possible key success factors that affect the effectiveness of FA. METHODS: This study employed a retrospective design using mixed methods for data collection. Data in the semesters 1/2020 and 2/2020 of the Doctor of Pharmacy curriculum in a Thailand pharmacy school were used. Three sets of data were gathered, including the course information (e.g. FA methods, FA scores, and SA scores) from 38 records, self-reports from 326 students and 27 teachers, and 5 focus group discussions. The quantitative data were statistically analyzed using descriptive statistics and Pearson correlation, while the qualitative data were analyzed using a content analysis framework. RESULTS: The analysis revealed five main methods that were used for FA, including individual quizzes, individual reports, individual skill assessments, group presentations, and group reports. Of all 38 courses, 29 (76.32%) had significant correlations between FA and SA scores at p-values < 0.05. The individual FA score was related to the correlation coefficient of the courses (p-value = 0.007), but the group FA score was not related (p-value = 0.081). In addition, only the frequency of individual quiz had a significant effect on the correlation coefficient. Moreover, the key success factors which affected the effectiveness of FA were divided into six themes, including the appropriate method, an effective reflection, frequency of assessment, the appropriate score, the adequate support system, and teacher knowledge management. CONCLUSION: The subjects that used individual FA methods provided a significant correlation between FA and SA, while those who used group FA methods did not show a significant correlation. Additionally, the key success factors in this study were appropriate assessment methods, frequency of assessment, effective feedback, appropriate scoring, and a proper support system.

Catalytic and structural insights into a stereospecific and thermostable Class II aldolase HpaI from Acinetobacter baumannii.

Aldolases catalyze the reversible reactions of aldol condensation and cleavage and have strong potential for the synthesis of chiral compounds, widely used in pharmaceuticals. Here, we investigated a new Class II metal aldolase from the p-hydroxyphenylacetate degradation pathway in Acinetobacter baumannii, 4-hydroxy-2-keto-heptane-1,7-dioate aldolase (AbHpaI), which has various properties suitable for biocatalysis, including stereoselectivity/stereospecificity, broad aldehyde utilization, thermostability, and solvent tolerance. Notably, the use of Zn2+ by AbHpaI as a native cofactor is distinct from other enzymes in this class. AbHpaI can also use other metal ion (M2+) cofactors, except Ca2+, for catalysis. We found that Zn2+ yielded the highest enzyme complex thermostability (Tm of 87 °C) and solvent tolerance. All AbHpaI•M2+ complexes demonstrated preferential cleavage of (4R)-2-keto-3-deoxy-D-galactonate ((4R)-KDGal) over (4S)-2-keto-3-deoxy-D-gluconate ((4S)-KDGlu), with AbHpaI•Zn2+ displaying the highest R/S stereoselectivity ratio (sixfold higher than other M2+ cofactors). For the aldol condensation reaction, AbHpaI•M2+ only specifically forms (4R)-KDGal and not (4S)-KDGlu and preferentially catalyzes condensation rather than cleavage by ∼40-fold. Based on 11 X-ray structures of AbHpaI complexed with M2+ and ligands at 1.85 to 2.0 Å resolution, the data clearly indicate that the M2+ cofactors form an octahedral geometry with Glu151 and Asp177, pyruvate, and water molecules. Moreover, Arg72 in the Zn2+-bound form governs the stereoselectivity/stereospecificity of AbHpaI. X-ray structures also show that Ca2+ binds at the trimer interface via interaction with Asp51. Hence, we conclude that AbHpaI•Zn2+ is distinctive from its homologues in substrate stereospecificity, preference for aldol formation over cleavage, and protein robustness, and is attractive for biocatalytic applications.

Sugar-sweetened beverages consumption during Covid-19 pandemic among office workers in semi-urban area in southern Thailand: a cross-sectional study.

Background: During COVID-19 pandemic, office worker has spent more than 6-8 hours per day sitting for online working following social distancing policy. Considering the popularity of online ordering and home delivery services, sugar-sweetened beverages (SSB) consumption have increased. However, the link between the types SSB consumption and their BMI was less well documented. Objective: To determine the association of the habitual intake (type, frequency, and volume) of sugar-sweetened beverages (SSB) with body mass index (BMI). Material and methods: A cross-sectional study, 337 office workers were selected according to probability proportionto-size and systematic random sampling. Data were collected using face-to-face interviews on the type, frequency, and volume of sugar-sweetened beverage intake. Samples of sugar-containing beverages were analyzed using high-throughput liquid chromatography/mass spectrometry (LC-MS/MS). The chi-square test was used to determine the relationship of SSB consumption with BMI. Unadjusted binary logistic regression analysis was used to assess the associations between BMI and metabolic diseases. Results: Most respondents (56.1%) were overweight (BMI >23 kg/m2). The most consumed SSB was milk tea (e.g., Thai tea and green tea), which was significantly related with BMI (p=0.03). LC-MS/MS analysis showed that sucrose and lactose were the major sugars in milk tea (34.7 g/100mL, on average). 70.6% of the respondents consumed >24 g/day of sugar, which is more than the World Health Organization's recommendation. Conclusions: Health control policies and health education, for example warning labels for the reduction of SSB consumption, may urgently be required to promote health in workplaces and prevent SSB-related metabolic diseases.

The Mechanism of Sugar C-H Bond Oxidation by a Flavoprotein Oxidase Occurs by a Hydride Transfer Before Proton Abstraction.

Understanding the reaction mechanism underlying the functionalization of C-H bonds by an enzymatic process is one of the most challenging issues in catalysis. Here, combined approaches using density functional theory (DFT) analysis and transient kinetics were employed to investigate the reaction mechanism of C-H bond oxidation in d-glucose, catalyzed by the enzyme pyranose 2-oxidase (P2O). Unlike the mechanisms that have been conventionally proposed, our findings show that the first step of the C-H bond oxidation reaction is a hydride transfer from the C2 position of d-glucose to N5 of the flavin to generate a protonated ketone sugar intermediate. The proton is then transferred from the protonated ketone intermediate to a conserved residue, His548. The results show for the first time how specific interactions around the sugar binding site promote the hydride transfer and formation of the protonated ketone intermediate. The DFT results are also consistent with experimental results including the enthalpy of activation obtained from Eyring plots, as well as the results of kinetic isotope effect and site-directed mutagenesis studies. The mechanistic model obtained from this work may also be relevant to other reactions of various flavoenzyme oxidases that are generally used as biocatalysts in biotechnology applications.

One-Pot Bioconversion of l-Arabinose to l-Ribulose in an Enzymatic Cascade.

This work reports the one-pot enzymatic cascade that completely converts l-arabinose to l-ribulose using four reactions catalyzed by pyranose 2-oxidase (P2O), xylose reductase, formate dehydrogenase, and catalase. As wild-type P2O is specific for the oxidation of six-carbon sugars, a pool of P2O variants was generated based on rational design to change the specificity of the enzyme towards the oxidation of l-arabinose at the C2-position. The variant T169G was identified as the best candidate, and this had an approximately 40-fold higher rate constant for the flavin reduction (sugar oxidation) step, as compared to the wild-type enzyme. Computational calculations using quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) showed that this improvement is due to a decrease in the steric effects at the axial C4-OH of l-arabinose, which allows a reduction in the distance between the C2-H and flavin N5, facilitating hydride transfer and enabling flavin reduction.

In vivo antimalarial effect of 1-hydroxy-5,6,7-trimethoxyxanthone isolated from Mammea siamensis T. Anders. flowers: pharmacokinetic and acute toxicity studies.

BACKGROUND: The potent antiplasmodial activity of 1-hydroxy-5,6,7-trimethoxyxanthone (HTX), isolated from Mammea siamensis T. Anders. flowers, has previously been demonstrated in vitro. However, its in vivo activity has not been reported. Therefore, this study aimed to investigate the antimalarial activity and acute toxicity of HTX in a mouse model and to evaluate the pharmacokinetic profile of HTX following a single intraperitoneal administration. METHODS: The in vivo antimalarial activity of HTX was evaluated using a 4-day suppressive test. Mice were intraperitoneally injected with Plasmodium berghei ANKA strain and given HTX daily for 4 days. To detect acute toxicity, mice received a single dose of HTX and were observed for 14 days. Additionally, the biochemical parameters of the liver and kidney functions as well as the histopathology of liver and kidney tissues were examined. HTX pharmacokinetics after intraperitoneal administration was also investigated in a mouse model. Liquid chromatography triple quadrupole mass spectrometry was used to quantify plasma HTX and calculate pharmacokinetic parameters with the PKSolver software. RESULTS: HTX at 10 mg/kg body weight significantly suppressed parasitemia in malaria-infected mice by 74.26%. Mice treated with 3 mg/kg HTX showed 46.88% suppression, whereas mice treated with 1 mg/kg displayed 34.56% suppression. Additionally, no symptoms of acute toxicity were observed in the HTX-treated groups. There were no significant alterations in the biochemical parameters of the liver and kidney functions and no histological changes in liver or kidney tissues. Following intraperitoneal HTX administration, the pharmacokinetic profile exhibited a maximum concentration (Cmax) of 94.02 ng/mL, time to attain Cmax (Tmax) of 0.5 h, mean resident time of 14.80 h, and elimination half-life of 13.88 h. CONCLUSIONS: HTX has in vivo antimalarial properties against P. berghei infection. Acute toxicity studies of HTX did not show behavioral changes or mortality. The median lethal dose was greater than 50 mg/kg body weight. Pharmacokinetic studies showed that HTX has a long elimination half-life; hence, shortening the duration of malaria treatment may be required to minimize toxicity.

The potency of HPLC-DAD and LC-MS/MS combined with ion chromatography for detection/purification of levulinic acid and bio-compounds from acid hydrolysis of OPEFB.

This work reports a new strategy for the detection and purification of levulinic acid (LA) and bio-compounds from the acid hydrolysis and enzymatic treatment of oil palm empty fruit bunch (OPEFB) through high-performance liquid chromatography (HPLC) techniques combined with ion/ligand chromatography. The detections of LA, biomass-saccharides, hydroxymethylfurfural (HMF), and furfural were successfully elucidated by optimizing the multiple reaction monitoring mode (MRM) and liquid chromatography conditions using a Pb2+ ligand exchange column in the liquid chromatography with tandem mass spectrometry (LC-MS/MS) approach. High-performance liquid chromatography with diode-array detection (HPLC-DAD) combined with an H+ ion exchange column also showed potency for detecting chromophoric compounds such as LA, HMF, furfural, and acid (by-products) but not biomass-saccharides. Both techniques showed acceptable validation in terms of linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, and stability in both quantitative and qualitative analysis. However, the LC-MS/MS approach showed higher sensitivity for detecting LA and HMF compared with HPLC-DAD. Samples comprised of cellobiose, glucose, HMF, and LA from the acid hydrolysis of cellulose to LA with a mineral acid, and the biocatalysis of cellulase and ß-glucosidase catalyzed cellulose (from OPEFB) to glucose were successfully monitored through the LC-MS/MS approach. In addition, using the optimal HPLC conditions obtained from LC-MS/MS, the purification of LA from other substances obtained from the hydrolysis reaction of cellulose (5 g) was successfully demonstrated by HPLC-DAD equipped with a fraction collector combined with an H+ ion exchange column at gram-scale of 1 g LA with a purification rate of 0.63 g ml-1 min-1.

Extraction of curcuminoids and ar-turmerone from turmeric (Curcuma longa L.) using hydrophobic deep eutectic solvents (HDESs) and application as HDES-based microemulsions.

The extraction of curcuminoids and aromatic (ar)-turmerone from Curcuma longa L. using organic solvents produces chemical waste, and is therefore incompatible with food applications. To address this issue, this study presents the design of hydrophobic deep eutectic solvents (HDESs) and HDES-based microemulsions. Using the response surface methodology (RSM), the optimal extraction conditions were identified as follows: HDES = OA:menthol (1:3.6 M ratio), solid-to-liquid ratio = 10:1 (mg/mL), and extraction duration = 90 min (prediction accuracy ≥ 85 %). Under these conditions, the HDES extraction yields of bisdemethoxycurcumin, demethoxycurcumin, curcumin, and ar-turmerone were 2.49 ± 0.25, 5.61 ± 0.45, 9.40 ± 0.86, and 3.83 ± 0.19 % (w/w, dry basis), respectively, while those obtained using the HDES-based microemulsion were 2.10 ± 0.18, 6.31 ± 0.48, 12.6 ± 1.20, and 2.58 ± 0.19 % (w/w, dry basis), respectively. The HDES and its microemulsions are more effective and environmentally friendly than conventional organic solvents for the extraction of curcuminoids and ar-turmerone, and these solvents are also compatible with food and pharmaceutical formulations.

Enzymatic fuel cells with an oxygen resistant variant of pyranose-2-oxidase as anode biocatalyst.

In enzymatic fuel cells (EnFCs), hydrogen peroxide formation is one of the main problems when enzymes, such as, glucose oxidase (GOx) is used due to the conversion of oxygen to hydrogen peroxide in the catalytic reaction. To address this problem, we here report the first demonstration of an EnFC using a variant of pyranose-2-oxidase (P2O-T169G) which has been shown to have low activity towards oxygen. A simple and biocompatible immobilisation approach incorporating multi-walled-carbon nanotubes within ferrocene (Fc)-Nafion film was implemented to construct EnFCs. Successful immobilisation of the enzymes was demonstrated showing 3.2 and 1.7-fold higher current than when P2O-T169G and GOx were used in solution, respectively. P2O-T169G showed 25% higher power output (maximum power density value of 8.45 ± 1.6 µW cm-2) and better stability than GOx in aerated glucose solutions. P2O-T169G maintained > 70% of its initial current whereas GOx lost activity > 90% during the first hour of 12 h operation at 0.15 V (vs Ag/Ag+). A different fuel cell configuration using gas-diffusion cathode and carbon paper electrodes were used to improve the power output of the fuel cell to 29.8 ± 6.1 µW cm-2. This study suggests that P2O-T169G with low oxygen activity could be a promising anode biocatalyst for EnFC applications.