Genomic insight into O-demethylation of 4-methoxybenzoate by a two-component system from Amycolatopsis magusensis KCCM40447.

Cytochrome P450 monooxygenases perform a multitude of roles, including the generation of hydroxylated aromatic compounds that might be utilized by microorganisms for their survival. WGS data of Amycolatopsis magusensis KCCM40447 revealed a complete circular genome of 9,099,986 base pairs and functionally assigned 8601 protein-encoding genes. Genomic analysis confirmed that the gene for 4-methoxybenzoate monoxygenase (CYP199A35) was conserved in close proximity to the gene for 4-hydroxybenzoate transporter (PcaK). The co-localized genes encoding CYP199A35, and ferredoxin-NAD(P) reductase (Mbr) represent a two-component system for electron transfer. CYP199A35 was specific for O-demethylation of para O-methyl substituted benzoic acid derivatives, 4-methoxybenzoate (4 MB), and 4-methoxycinnamic acid (4MCA) using the native redox partner (Mbr); two-component system and non-physiological redox partners (Pdr/Pdx); three-component system. The catalytic efficiency for O-demethylation of 4 MB using Mbr and Pdr/Pdx was 0.02 ± 0.006 min-1 µM-1 and 0.07 ± 0.02 min-1 µM-1 respectively. Further, sequence annotation and function prediction by RAST and KEEG analysis revealed a complete catabolic pathway for the utilization of 4 MB by strain KCCM40447, which was also proved experimentally.

Analysis of critical residues for peroxygenation and improved peroxygenase activity via in situ H2O2 generation in CYP105D18.

Limited numbers of CYPs have been reported to work naturally as peroxygenases. The peroxide shunt pathway can be efficiently used as an alternative for the NAD(P)H and reductase systems, particularly in high hydrogen peroxide (H2O2) resistance CYPs. We reported the structural and biochemical features of CYP105D18 peroxygenase for its high H2O2 tolerance capacity. Q348 was a crucial residue for the stability of CYP105D18 during the exposure to H2O2. In addition, the role of the hydrophilic amino acid T239 from the I helix for peroxygenation and regiospecificity toward testosterone was investigated. Interestingly, T239E differs in product formation from wild type, catalyzing testosterone to androstenedione in the presence of H2O2. The other variant, T239A, worked with the Pdx/Pdr system and was unable to catalyze testosterone conversion in the presence of H2O2, suggesting the transformation of peroxygenase into monooxygenase. CYP105D18 supported the alternative method of H2O2 used for the catalysis of testosterone. The use of the same concentration of urea hydrogen peroxide adducts in place of direct H2O2 was more efficient for 2ß-hydroxytestosterone conversion. Furthermore, in situ H2O2 generation using GOx/glucose system enhanced the catalytic efficiency (kcat/Km) for wild type and F184A by 1.3- and 1.9-fold, respectively, compared to direct use of H2O2 The engineering of CYP105D18, its improved peroxygenase activity, and alteration in the product oxidation facilitate CYP105D18 as a potential candidate for biotechnological applications.

Biochemical and structural basis of mercuric reductase, GbsMerA, from Gelidibacter salicanalis PAMC21136.

Heavy metals, including mercury, are non-biodegradable and highly toxic to microorganisms even at low concentrations. Understanding the mechanisms underlying the environmental adaptability of microorganisms with Hg resistance holds promise for their use in Hg bioremediation. We characterized GbsMerA, a mercury reductase belonging to the mercury-resistant operon of Gelidibacter salicanalis PAMC21136, and found its maximum activity of 474.7 µmol/min/mg in reducing Hg+2. In the presence of Ag and Mn, the enzyme exhibited moderate activity as 236.5 µmol/min/mg and 69 µmol/min/mg, respectively. GbsMerA exhibited optimal activity at pH 7.0 and a temperature of 60 °C. Moreover, the crystal structure of GbsMerA and structural comparison with homologues indicated that GbsMerA contains residues, Tyr437´ and Asp47, which may be responsible for metal transfer at the si-face by providing a hydroxyl group (-OH) to abstract a proton from the thiol group of cysteine. The complex structure with NADPH indicated that Y174 in the re-face can change its side chain direction upon NADPH binding, indicating that Y174 may have a role as a gate for NADPH binding. Moreover, the heterologous host expressing GbsMerA (pGbsMerA) is more resistant to Hg toxicity when compared to the host lacking GbsMerA. Overall, this study provides a background for understanding the catalytic mechanism and Hg detoxification by GbsMerA and suggests the application of genetically engineered E. coli strains for environmental Hg removal.

Identification and evaluation of CAZyme genes, along with functional characterization of a new GH46 chitosanase from Streptomyces sp. KCCM12257.

The genomic analysis of Streptomyces sp. KCCM12257 presented 233 CAZyme genes with a predominant glycosyl hydrolase family. This contributes degradation of various polysaccharides including chitin and chitosan, and other promising candidates for the production of different oligosaccharides. We screened the strain providing different polysaccharides as a sole source of carbon and strain KCCM12257, showed higher activity towards colloidal chitosan. Further, we identified and characterized a new chitosanase (MDI5907146) of GH46 family. There was no activity towards chitin, carboxymethylcellulose, or even with chitosan powder. This enzyme acts on colloidal chitosan and hydrolyzes it down into monoacetyl chitobiose, which consists of two glucosamine units with an acetyl group attached to them. The maximum enzyme activity was observed at pH 6.5 and 40 °C using colloidal chitosan as a substrate. The Co2+ metal ions almost double the reaction as compared to other metal ions. The dissociation constant (Km) and of colloidal chitosan (≥90 % and ≥75%DD) were 3.03 mg/ml and 5.01 mg/ml respectively, while maximum velocity (Vmax) values were found to be 36 mg/ml, and 30 µM/µg/min, respectively. Similarly, catalytic efficiency (Kcat/Km) of colloidal chitosan with ≥90 %DD was 1.9 fold higher than colloidal chitosan with ≥75%DD.

H2O2-Driven Hydroxylation of Steroids Catalyzed by Cytochrome P450 CYP105D18: Exploration of the Substrate Access Channel.

CYP105D18 supports H2O2 as an oxygen surrogate for catalysis well and shows high H2O2 resistance capacity. We report the hydroxylation of different steroids using H2O2 as a cosubstrate. Testosterone was regiospecifically hydroxylated to 2ß-hydroxytestosterone. Based on the experimental data and molecular docking, we predicted that hydroxylation of methyl testosterone and nandrolone would occur at position 2 in the A-ring, while hydroxylation of androstenedione and adrenosterone was predicted to occur in the B-ring. Further, structure-guided rational design of the substrate access channel was performed with the mutagenesis of residues S63, R82, and F184. Among the mutants, S63A showed a marked decrease in product formation, while F184A showed a significant increase in product formation in testosterone, nandrolone, methyl testosterone, androstenedione, and adrenosterone. The catalytic efficiency (kcat/Km) toward testosterone was increased 1.36-fold in the F184A mutant over that in the wild-type enzyme. These findings might facilitate the potential use of CYP105D18 and further engineering to establish the basis of biotechnological applications. IMPORTANCE The structural modification of steroids is a challenging chemical reaction. Modifying the core ring and the side chain improves the biological activity of steroids. In particular, bacterial cytochrome P450s are used as promiscuous enzymes for the activation of nonreactive carbons of steroids. In the present work, we reported the H2O2-mediated hydroxylation of steroids by CYP105D18, which also overcomes the use of expensive cofactors. Further, exploring the substrate access channel and modifying the bulky amino acid F184A increase substrate conversion while modifying the substrate recognizing amino acid S63 markedly decreases product formation. Exploring the substrate access channel and the rational design of CYP105D18 can improve the substrate conversion, which facilitates the engineering of P450s for industrial application.

Implication of Salivary Biochemical Parameters for Diagnosis and Prognosis of Type 2 Diabetes Mellitus.

Background: Clinical laboratory diagnosis and prognosis for diabetes mellitus is performed using blood as a major specimen; however, saliva may represent as an alternative noninvasive specimen of choice. This study aims to evaluate salivary biochemical parameters in diabetic and healthy individuals to substantiate saliva's role in the diagnosis and prognosis of type 2 diabetes mellitus (T2DM). Methods: This case-control study included 150 T2DM patients and 150 apparently healthy individuals. Socio-demographic data and anthropometric measurements were recorded using a standard questionnaire. Correlation between salivary and blood levels for each parameter was determined using Pearson correlation. Linear regression was performed to estimate the blood levels of the parameters from their salivary levels. Receiver operating characteristics (ROC) analysis was done to determine the diagnostic ability of salivary glucose and establish a sensitivity, specificity, and cut-off value. Results: Salivary glucose, TC, LDL-C, urea, and creatinine were significantly higher in people with diabetes than in the control population (p < 0.05). A significant positive correlation was found between salivary and blood parameters including glucose, TC, TG, LDL-C, urea, and creatinine except for HDL-C in both case and control groups. The linear relationship for each parameter, except glucose in case population and HDL-C in case, control, and the total population was observed between blood and saliva. ROC analysis gave a cut-off value of 1.9 mg/dl for salivary glucose with 71.4% sensitivity and 72.3% specificity. Conclusion: Salivary estimation significantly reflects the blood parameters in this study, indicating that saliva can be a noninvasive specimen for the diagnosis and prognosis of T2DM.

Crystal structure of ChbG from Klebsiella pneumoniae reveals the molecular basis of diacetylchitobiose deacetylation.

The chitobiose (chb) operon is involved in the synthesis of chitooligosaccharide and is comprised of a BCARFG gene cluster. ChbG encodes a chitooligosaccharide deacetylase (CDA) which catalyzes the removal of one acetyl group from N,N'-diacetylchitobiose. It is considered a novel type of CDA due to its lack of sequence homology. Although there are various structural studies of CDAs linked to the kinetic properties of the enzyme, the structural information of ChbG is unavailable. In this study, the crystal structure of ChbG from Klebsiella pneumoniae is provided. The molecular basis of deacetylation of diacetylchitobiose by ChbG is determined based on structural analysis, mutagenesis, biophysical analysis, and in silico docking of the substrate, diacetylchitobiose. This study contributes towards a deeper understanding of chitin and chitosan biology, as well as provides a platform to engineer CDA biocatalysts.

Characterization of high-H2O2-tolerant bacterial cytochrome P450 CYP105D18: insights into papaverine N-oxidation.

The bacterial CYP105 family is involved in secondary metabolite biosynthetic pathways and plays essential roles in the biotransformation of xenobiotics. This study investigates the newly identified H2O2-mediated CYP105D18 from Streptomyces laurentii as the first bacterial CYP for N-oxidation. The catalytic efficiency of CYP105D18 for papaverine N-oxidation was 1.43 s-1 µM -1. The heme oxidation rate (k) was low (<0.3 min-1) in the presence of 200 mM H2O2. This high H2O2 tolerance capacity of CYP105D18 led to higher turnover prior to heme oxidation. Additionally, the high-resolution papaverine complexed structure and substrate-free structure of CYP105D18 were determined. Structural analysis and activity assay results revealed that CYP105D18 had a strong substrate preference for papaverine because of its bendable structure. These findings establish a basis for biotechnological applications of CYP105D18 in the pharmaceutical and medicinal industries.

Crystal structure of an apo 7α-hydroxysteroid dehydrogenase reveals key structural changes induced by substrate and co-factor binding.

7α-Hydroxysteroid dehydrogenase (7α-HSDH) catalyzes the dehydrogenation of a hydroxyl group at the 7α position in steroid substrates using NAD+ or NADP+ as a co-factor. Although studies have determined the binary and ternary complex structures, detailed structural changes induced by ligand and co-factor binding remain unclear, because ligand-free structures are not yet available. Here, we present the crystal structure of apo 7α-HSDH from Escherichia coli (Eco-7α-HSDH) at 2.7 Å resolution. We found that the apo form undergoes substantial conformational changes in the ß4-α4 loop, α7-α8 helices, and C-terminus loop among the four subunits comprising the tetramer. Furthermore, a comparison of the apo structure with the binary (NAD+)-complex and ternary (NADH and 7-oxoglycochenodeoxycholic acid)-complex Eco-7α-HSDH structures revealed that only the ternary-complex structure has a fully closed conformation, whereas the binary-complex and apo structures have a semi-closed or open conformation. This open-to-closed transition forces several catalytically important residues (S146, Y159, and K163) into correct positions for catalysis. To confirm the catalytic activity, we used alcohol dehydrogenase for NAD+ regeneration to allow efficient conversion of chenodeoxycholic acid to 7-ketolithocholic acid by Eco-7α-HSDH. These findings demonstrate that apo Eco-7α-HSDH exhibits intrinsically flexible characteristics with an open conformation. This structural information provides novel insight into the 7α-HSDH reaction mechanism.

Vitamin D deficiency and cardiovascular risk in type 2 diabetes population.

This study aims to assess vitamin D deficiency-induced dyslipidemia and cardiovascular disease (CVD) risk in poor glycemic control among type 2 diabetes mellitus (T2DM) patients. This study was carried out among 455 T2DM patients involving poor glycemic control (n = 247) and good glycemic control (n = 208). Fasting plasma glucose (FPG) and HbA1c were measured to assess glycemic control. Cardiac risk ratio, atherogenic index plasma, and atherogenic coefficient were calculated to assess and compare the CVD risk in different groups. Patients with poor control had a significantly higher level of total cholesterol (TC), triglyceride (TG), and non-high-density lipoprotein lipase cholesterol (non-HDL-C), atherogenic variables, and lower level of high-density lipoprotein lipase cholesterol (HDL-C) as compared to patients with good glycemic control. We also observed significant negative correlation of vitamin D with lipid markers and atherogenic variables in poor glycemic control diabetic population. The serum vitamin D levels were inversely associated with HbA1c, FPG, TG, TC, and non-HDL-C. Furthermore, hypercholesterolemia, hypertriglyceridemia, and elevated non-HDL-C were the independent risks in hypovitaminosis D population. Vitamin D deficiency in poor glycemic control is likely to develop dyslipidemia as compared to vitamin D insufficient and sufficient groups. Thus, vitamin D supplementation and an increase in exposure to sunlight may reduce the risk of cardiovascular complications in diabetes.

Metabolic Syndrome and Diabetes Risk Among Young Adult Students in the Health Sciences from Kathmandu, Nepal.

BACKGROUND: The widespread dissemination of unhealthy dietary habits, childhood-teenage obesity, and sedentary lifestyle in young adults has paved the way for public health burden metabolic syndrome and early onset of type 2 diabetes mellitus. The aim of this study was to assess the prevalence and risk factors for metabolic syndrome and diabetes among young adult students. METHODS: This cross-sectional study was conducted among students of age group (18 to 25 years) studying at Manmohan Memorial Institute of Health Sciences and Central Institute of Science and Technology. The diabetes risk score of each individual was calculated by the Finnish Diabetes Risk Score (FINDRISC tool). Independent risk factors for diabetes and metabolic syndrome were measured by multivariable logistic regression analysis. The p-value of <0.05 was considered statistically significant in this study. RESULTS: A total of 825 students were recruited and 739 (89.6%) students completed the study with all the fulfilled criteria. The metabolic syndrome (Harmonized Joint Scientific Statement (HJSS) criteria) was present in 7.1%, and the most prevalent defining component was low HDL-C (78%); 74.8% of students were under low risk, 22.18% were at slightly elevated risk, 2.02% were at moderate risk, and 1.01% were at high risk of diabetes. The cardiometabolic risk factors like BMI, TC, and LDL-C were higher at a significant level (p<0.001) with an increased diabetes risk score. Independent lifestyle risk factor for metabolic syndrome was current smoking (AOR, 4.49, 95% CI 1.38-14.62) whereas, an independent lifestyle risk factor for diabetes was low adherence to physical exercise (AOR, 4.81, 95% CI, 2.90-7.99). CONCLUSION: Metabolic syndrome is present, although in low numbers in young adults putting them at risk to develop diabetes in the near future. Early assessment of metabolic syndrome and diabetes risk in young may provide insights for preventive and control plans for risk population.

Classical Dermatomyositis: A Case Report.

Dermatomyositis is an idiopathic myopathy involving progressive muscle weakness with skin manifestation. Diagnosis is based upon the progressive muscle weakness, skin rashes, elevated serum muscle enzymes, muscle biopsy, and abnormal electromyogram. The incidence of dermatomyositis is rare. In this case study, we report a case of classical dermatomyositis without incidence of malignancy. Timely diagnosis and administration of steroid led to better prognosis of the patient.

Self-Care Adherence And Barriers To Good Glycaemic Control In Nepalese Type 2 Diabetes Mellitus Patients: A Hospital-Based Cross-Sectional Study.

PURPOSE: The patient believes in adherence to medication rather than to self-care adherence and lifestyle changes for the management of diabetes. This study was carried out to establish the association of self-care adherence and their barriers in poor glycemic control in our diabetic population. PATIENTS AND METHODS: This cross-sectional study was conducted among 480 already diagnosed diabetes outpatients attended in our two hospitals. Glycaemic control was defined by levels of HbA1c. Socio-demographic data, lifestyle variables and anthropometric measurements were recorded using a standard questionnaire. Fasting blood glucose, HbA1c and lipid profiles were estimated using the manufacturer's guideline. Student's t-test and one-way ANOVA were used for comparison between different groups and the correlation was established by Spearman correlation. Risk factors associated with poor glycaemic control were verified by logistic regression analysis. RESULTS: The mean HbA1c of the study population was 7.4±1.3% and 65.4% had poor glycaemic control with mean 8.0±1.1%. Higher HbA1c levels were significantly associated with duration of diabetes, a number of drugs used, patient-physician relationship and knowledge about diabetes. The poor glycaemic control was significantly associated with low adherence of following the meal plan, regular medication and regular exercising (p<0.001). Among all the barriers, a too busy schedule for following the meal plan, taking medications and exercising regularly was significantly correlated with HbA1c levels. Multivariable logistic regression analysis showed irregular meal plan (OR=5.27), irregular exercise (OR=2.25), number of medication used (OR= 0.19) and lesser extent patient-physician relationship (OR=2.68) were independent risk factors for poor glycaemic control. CONCLUSION: The poor glycaemic control was associated with poor adherence to self-care adherence and their barriers in our diabetic population. Integrated knowledge on diabetes management should be targeted to improve glycaemic control in our communities.

Metabolic syndrome and biochemical changes among non-alcoholic fatty liver disease patients attending a tertiary care hospital of Nepal.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is mutually and bidirectionally linked with metabolic syndrome (MetS) of which it is both the cause and the consequences. Worldwide, 6.3 to 33% of the general populations are estimated to suffer from the disease with even higher prevalence in the group sharing metabolic co-morbidities. Hence, this study aims to recognize various risk factors including metabolic components and blood parameters to predict the possible incidence of the disease. METHODS: Total of 429 (219 NAFLD and 210 control) subjects were conveniently selected for study during the period of 9 months. Diagnosis of non-alcoholic fatty liver disease was done by liver imaging and based on liver enzymes. Assessment of metabolic syndrome was done by International Diabetic Federation (IDF) and National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) criteria. All biochemical and hematological parameters and liver enzymes were estimated by using standard guideline. Mean comparison of quantitative data in different groups were performed using analysis of variance (one-way ANOVA). Risk estimation of NAFLD associated with each character was verified by Chi-square test. RESULTS: There was significant high levels of body mass index (BMI), waist circumference (WC) and lipid profiles in NAFLD patients in comparison to control population (p < 0.001). Further, according to the NCEP ATP III criteria, 13.6% of NAFLD were present with MetS where risk estimate was significant (OR = 2.15). Whereas, other criteria (IDF) for MetS showed higher frequency (30.1%) with higher risk (OR = 29.75) for the presence of MetS in NAFLD patients. The change in triglycerides (TG) and HDL-C (high density lipoprotein cholesterol) was also statistically significant in different grades of NAFLD. High risk for NAFLD was associated with existing co-morbid conditions like cardiovascular risk patients (3.18 times) followed by obese patients (1.72 times) and Diabetes Mellitus patients (1.68 times) at a significant level. CONCLUSION: The result of this study suggests that there is an increased prevalence of all the components of MetS and significant changes in biochemical markers in cases of NAFLD. Timely diagnosis would help in delaying its complications and co-morbidities.

Effect of age and estrogen on biochemical markers of bone turnover in postmenopausal women: a population-based study from Nepal.

BACKGROUND: Osteoporosis, a common disease worldwide, is characterized by low bone mass and architectural deterioration of bone tissue, leading to enhanced bone fragility and increase in fracture risks. Both menopause and aging are related factors leading to greater risk of bone disease, particularly among postmenopausal women. Hence, the main purpose of this study was to investigate the differences in biochemical markers of bone turnover and to evaluate the association of hormones and age-related factors with biochemical markers between pre- and postmenopausal women. METHODS: A descriptive cross-sectional study was conducted over a period of 6 months among the female population of Dholahity community, Lalitpur, Nepal. A total of 496 healthy women were selected based on the questionnaire strategy. Among them, 244 were premenopausal and 252 were postmenopausal women. Different bone markers were evaluated as per the guideline provided by the reagent manufacturer, and hormonal assay, particularly estradiol level assessment, was performed by chemiluminescence immunoassay-based technique. RESULTS: A significant decrease in serum calcium level and estradiol level was observed in postmenopausal women as compared to premenopausal women, whereas a significant increase in serum phosphorus and alkaline phosphatase (ALP) levels was seen among postmenopausal (p<0.001). Age was significantly correlated with bone markers (ALP and calcium) in postmenopausal group (p<0.005), while there was no significant correlation in premenopausal group. In addition, there was significant positive correlation between calcium and estradiol in postmenopausal women, while ALP was negatively correlated with estradiol in that group. Further, no significant correlation was demonstrated between estradiol and bone markers in postmenopausal women in body mass index and age-adjusted partial correlation analysis. CONCLUSION: Timely diagnosis of osteoporosis in women would be of significant benefit for effective care for required populations and help to minimize mortality rate and financial burden of our country.

Elevated Cardiovascular Risks among Postmenopausal Women: A Community Based Case Control Study from Nepal.

Cardiovascular disease (CVD) is one of the leading causes of death worldwide which is more prevalent in women after menopause. Hormonal changes associated with menopause are accountable for dyslipidemic pattern that causes CVD and associated complications. Therefore, the present study was commenced to compare the lipid profile in pre- and postmenopausal women. A descriptive cross-sectional study was conducted at Manmohan Memorial Institute of Health Sciences (MMIHS) from February 2016 to July 2016. A total of 260 fasting samples were collected from healthy women, 130 from premenopausal and 130 from postmenopausal women, and analyzed for Total Cholesterol (TC), Triacylglycerol (TAG), High Density Lipoprotein Cholesterol (HDL-C), and Low Density Lipoprotein Cholesterol (LDL-C) as per the guideline provided by the reagent manufacturer (Human, Germany). All the parameters were analyzed by Stat Fax 3300 semi auto analyzer. TC, TAG, HDL-C, and LDL-C were highly significantly increased in postmenopausal women when compared to premenopausal women. LDL/HDL ratio was significantly elevated in postmenopausal women than in premenopausal women. BMI was significantly positively correlated with TC and TAG in both pre- and postmenopausal population and it was positively correlated with HDL-C in premenopausal population while negatively correlated in postmenopausal population. Since more of the atherogenic lipid parameters are increased in postmenopausal women, they appear to be more prone to have CVD and associated complications in near future. Hence, it is mandatory to monitor and manage dyslipidemic pattern in every woman experiencing menopause.

Shigellosis Caused by CTX-M Type ESBL Producing Shigella flexneri in Two Siblings of Rural Nepal: First Case Report from the Country.

Shigellosis is an acute infectious disease characterized as severe bloody diarrhea (dysentery) and is accountable for a significant burden of morbidity and mortality especially in children under the age of 5 years. Antimicrobial therapy is required in the cases of severe dysentery associated with Shigella. However, emergence of multidrug resistant (MDR) strains of Shigella spp. over the last two decades has restricted the use of common therapeutic antimicrobials. In MDR strains, the third-generation cephalosporins have been used for the treatment, but, unfortunately, emerging reports of enzyme mediated ß-lactam resistance among Shigella isolates from various parts of the world have greatly compromised the therapy of pediatric dysentery. In Nepal, drug resistant strains of Shigella spp. have been reported, but MDR and extended spectrum ß-lactamase (ESBL) producing strains were previously unknown. Here, we report two Shigella flexneri isolates harboring ESBL genotype-CTX-M associated with acute dysentery in two siblings which were presented and treated in a tertiary care teaching hospital of Kathmandu, Nepal.