Mechanisms of enzymes of the a-aminoadipate pathway: Homocitrate synthase from Thermus thermophilus and saccharopine dehydrogenase from Saccharomyces cerevisiae

The &alpha-aminoadipate (AAA) pathway for lysine biosynthesis is nearly unique to higher fungi, including human and plant pathogens and euglenoids; an exception is the thermophilic bacterium Thermus thermophilus. . Knock-out of the genes in this pathway has been shown to be lethal in Saccharomyces cerevisiae. It has been shown that scavenging of lysine is insufficient for survival in the host. Thus, enzymes of this pathway could be potential drug targets. The AAA pathway is comprised of eight enzymatic reactions catalyzed by seven enzymes. Homocitrate synthase (HCS) catalyzes the first and regulated step in this pathway, the condensation of acetyl-CoA (AcCoA) and &alpha-ketoglutarate (&alpha-Kg) to give homocitrate and coenzyme A (CoASH). The homocitrate synthase from Thermus thermophilus (TtHCS) is a metal activated enzyme with either Mg2+ or Mn2+ capable of serving as the divalent cation. The enzyme exhibits a sequential kinetic mechanism. The mechanism is steady state ordered with &alpha-Kg binding prior to AcCoA with Mn2+, while it is steady state random with Mg2+, suggesting a difference in the competence of the E-Mn-&alpha-Kg-AcCoA and E-Mg-&alpha-Kg-AcCoA complexes. The mechanism is supported by product and dead-end inhibition studies. The primary isotope effect obtained with deuterioacetylCoA (AcCoA-d3) in the presence of Mg2+ is unity at low concentrations of AcCoA, while it is 2 at high concentrations of AcCoA. Data suggest the presence of a slow conformational change induced by binding of AcCoA that accompanies deprotonation of the methyl group of AcCoA. The solvent kinetic deuterium isotope effect is also unity at low AcCoA, but is 1.7 at high AcCoA, consistent with the proposed slow conformational change. The maximum rate is pH independent with either Mg2+ or Mn2+ as the divalent metal ion, while V/Ka-Kg (with Mn2+) decreases at low and high pH giving pK values of about 6.5 and 8.0. Lysine is a competitive inhibitor that binds to the active site of TtHCS, and shares some of the same binding determinants as &alpha-Kg. Lysine binding exhibits negative cooperativity, indicating crosstalk between the two monomers of the TtHCS dimer. Data are discussed in terms of the overall mechanism of TtHCS.Saccharopine dehydrogenase (SDH) catalyzes the final reaction in the &alpha-aminoadipate pathway, the conversion of L-saccharopine to L-lysine and &alpha-ketoglutarate using NAD+ as an oxidant. The enzyme utilizes a general acid-base mechanism to carry out the multistep saccharopine dehydrogenase reaction with a base proposed to accept a proton from the secondary amine in the oxidation step and a second group proposed to activate water to hydrolyze the imine. A pair of thiols in the dinucleotide binding site forms a disulfide in the wild type (WT) enzyme as isolated, which interferes with binding of the dinucleotide substrate. The SDH enzyme with a C205S mutation, has been characterized recently and is referred to as a pseudo-WT enzyme. Crystal structures of an open apo-form of the pseudo-WT SDH (C205S), as well as a closed form of the C205S enzyme with saccharopine and NADH bound have been solved. The structure of a ternary complex between the C205S pseudo-WT enzyme, NADH, and Sacc provided a closed form of the enzyme and a more accurate description of the interactions between enzyme side chains and reactant functional groups. Importantly, the distance between C4 of the nicotinamide ring to C8 of Sacc is 3.6 Å, a reasonable hydride transfer distance. The side chains of H96 and K77 now appear properly positioned to act as acid-base catalysts. Mutation of K77 to M results in a 145-fold decrease in V/Et and greater than a three order of magnitude increase in V2/KLysEt and V2/K&alpha-KgEt. A primary deuterium kinetic isotope effect of 2.0 and an inverse solvent deuterium isotope effect of 0.77 on V2/KLys were observed, suggesting that hydride transfer is rate-limiting. The hypothesis was corroborated by the value of 2.0 obtained when the primary deuterium kinetic isotope effect was repeated in D2O. The viscosity effect of 0.8 observed on V2/KLys indicated the solvent deuterium isotope effect resulted from stabilization of an enzyme form prior to hydride transfer. The deuterium isotope effect on V is slightly lower than that on V/K and decreases when repeated in D2O, suggesting a contribution to rate limitation of product release, likely release of NAD+. A small normal solvent isotope effect is observed on V, which decreases slightly when repeated with NADD, consistent with a contribution from product release to rate limitation. In addition, V2/KLysEt is pH independent consistent with the loss of an acid-base catalyst and perturbation of the pKa of the second catalytic group to higher pH, likely a result of a change in the overall charge in the active site. The H96Q mutation results in about a 28-fold decrease in V2/Et and >103-fold decreases in the second order rate constant. The primary deuterium kinetic isotope effect is within error 1, but a large solvent deuterium isotope effect of 2.4 is observed, suggesting rate limiting imine hydrolysis, consistent with the proposed role of H96 in protonating the leaving hydroxyl as the imine is formed. In agreement, the multiple isotope effects, repeating the primary deuterium effect in D2O and the solvent effect with NADD, are identical to the individual effect. The pH-rate profile for V2/KLysEt exhibits the pKa for K77, perturbed to a value of about 9, which must be unprotonated in order to accept a proton from the epsilon-amine of the substrate Lys so that it can act as a nucleophile. The proposed roles of H96 and K77 are corroborated by the nearly 700-fold decrease in V2/Et and >105-fold decreases in the second order rate constants for the double mutant. Thus, data consistent with an acid-base mechanism in the non-physiologic reaction direction suggest that the K77 side chain initially accepts a proton from the epsilon-amine of the substrate lysine and eventually donates it to the imino nitrogen as it is reduced to a secondary amine in the hydride transfer step, and then H96 protonates the carbonyl oxygen as the imine is formed.Lysine13, positioned near the active site base (K77), hydrogen-bonds to a glutamate neutralizing it, contributing to setting the pKa of the catalytic residues to near neutral pH. Glutamate16 hydrogen-bonds with N-epsilon of R18 which in turn has strong H-bonding interactions with &alpha-carboxylate of &alpha-Kg. Mutation of K13 to M and E16 to Q decreased kcat ~ 15-fold, and primary and solvent deuterium isotope effects measured with the mutant enzymes indicate hydride transfer is rate limiting of SDH reaction. The pH-rate profiles for K13 exhibited no pH dependence, consistent with an increase in negative charge in the active site resulting in the perturbation in the pKas of catalytic groups. Elimination of E16 affects optimal positioning of R18 for binding and holding &alpha-Kg in the correct conformation for optimum catalysis. As a result, the delta delta G°' value of 2.60 kcal/mol for E16 suggests its contribution in binding of Lys.Overall, data are consistent with the proposed acid-base mechanism in the non-physiologic reaction direction in which the K77 side chain initially accepts a proton from the &alpha-amine of the substrate lysine and eventually donates it to the imino nitrogen as it is reduced to a secondary amine in the hydride transfer step, and then H96 protonates the carbonyl oxygen as the imine is formed. Lysine13 and E16 play an important role of balancing the charge in the active site and elimination of either of the residues perturbs pKas of the catalytic residues, positioning of R18 residue for optimum binding of &alpha-Kg is affected and the step contributing to the rate-limitation is changed to hydride transfer while in the case of C205S enzyme neither hydride transfer nor imine hydrolysis was solely responsible for rate-limitation. Thus K13 and E16 are important for favorable binding of Lys and &alpha-Kg and optimum catalysis

The global, regional, and national burden of adult lip, oral, and pharyngeal cancer in 204 countries and territories:A systematic analysis for the Global Burden of Disease Study 2019

Importance Lip, oral, and pharyngeal cancers are important contributors to cancer burden worldwide, and a comprehensive evaluation of their burden globally, regionally, and nationally is crucial for effective policy planning.Objective To analyze the total and risk-attributable burden of lip and oral cavity cancer (LOC) and other pharyngeal cancer (OPC) for 204 countries and territories and by Socio-demographic Index (SDI) using 2019 Global Burden of Diseases, Injuries, and Risk Factors (GBD) Study estimates.Evidence Review The incidence, mortality, and disability-adjusted life years (DALYs) due to LOC and OPC from 1990 to 2019 were estimated using GBD 2019 methods. The GBD 2019 comparative risk assessment framework was used to estimate the proportion of deaths and DALYs for LOC and OPC attributable to smoking, tobacco, and alcohol consumption in 2019.Findings In 2019, 370 000 (95% uncertainty interval [UI], 338 000-401 000) cases and 199 000 (95% UI, 181 000-217 000) deaths for LOC and 167 000 (95% UI, 153 000-180 000) cases and 114 000 (95% UI, 103 000-126 000) deaths for OPC were estimated to occur globally, contributing 5.5 million (95% UI, 5.0-6.0 million) and 3.2 million (95% UI, 2.9-3.6 million) DALYs, respectively. From 1990 to 2019, low-middle and low SDI regions consistently showed the highest age-standardized mortality rates due to LOC and OPC, while the high SDI strata exhibited age-standardized incidence rates decreasing for LOC and increasing for OPC. Globally in 2019, smoking had the greatest contribution to risk-attributable OPC deaths for both sexes (55.8% [95% UI, 49.2%-62.0%] of all OPC deaths in male individuals and 17.4% [95% UI, 13.8%-21.2%] of all OPC deaths in female individuals). Smoking and alcohol both contributed to substantial LOC deaths globally among male individuals (42.3% [95% UI, 35.2%-48.6%] and 40.2% [95% UI, 33.3%-46.8%] of all risk-attributable cancer deaths, respectively), while chewing tobacco contributed to the greatest attributable LOC deaths among female individuals (27.6% [95% UI, 21.5%-33.8%]), driven by high risk-attributable burden in South and Southeast Asia.Conclusions and Relevance In this systematic analysis, disparities in LOC and OPC burden existed across the SDI spectrum, and a considerable percentage of burden was attributable to tobacco and alcohol use. These estimates can contribute to an understanding of the distribution and disparities in LOC and OPC burden globally and support cancer control planning efforts

Global burden of 288 causes of death and life expectancy decomposition in 204 countries and territories and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

BACKGROUND Regular, detailed reporting on population health by underlying cause of death is fundamental for public health decision making. Cause-specific estimates of mortality and the subsequent effects on life expectancy worldwide are valuable metrics to gauge progress in reducing mortality rates. These estimates are particularly important following large-scale mortality spikes, such as the COVID-19 pandemic. When systematically analysed, mortality rates and life expectancy allow comparisons of the consequences of causes of death globally and over time, providing a nuanced understanding of the effect of these causes on global populations. METHODS The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 cause-of-death analysis estimated mortality and years of life lost (YLLs) from 288 causes of death by age-sex-location-year in 204 countries and territories and 811 subnational locations for each year from 1990 until 2021. The analysis used 56 604 data sources, including data from vital registration and verbal autopsy as well as surveys, censuses, surveillance systems, and cancer registries, among others. As with previous GBD rounds, cause-specific death rates for most causes were estimated using the Cause of Death Ensemble model-a modelling tool developed for GBD to assess the out-of-sample predictive validity of different statistical models and covariate permutations and combine those results to produce cause-specific mortality estimates-with alternative strategies adapted to model causes with insufficient data, substantial changes in reporting over the study period, or unusual epidemiology. YLLs were computed as the product of the number of deaths for each cause-age-sex-location-year and the standard life expectancy at each age. As part of the modelling process, uncertainty intervals (UIs) were generated using the 2·5th and 97·5th percentiles from a 1000-draw distribution for each metric. We decomposed life expectancy by cause of death, location, and year to show cause-specific effects on life expectancy from 1990 to 2021. We also used the coefficient of variation and the fraction of population affected by 90% of deaths to highlight concentrations of mortality. Findings are reported in counts and age-standardised rates. Methodological improvements for cause-of-death estimates in GBD 2021 include the expansion of under-5-years age group to include four new age groups, enhanced methods to account for stochastic variation of sparse data, and the inclusion of COVID-19 and other pandemic-related mortality-which includes excess mortality associated with the pandemic, excluding COVID-19, lower respiratory infections, measles, malaria, and pertussis. For this analysis, 199 new country-years of vital registration cause-of-death data, 5 country-years of surveillance data, 21 country-years of verbal autopsy data, and 94 country-years of other data types were added to those used in previous GBD rounds. FINDINGS The leading causes of age-standardised deaths globally were the same in 2019 as they were in 1990; in descending order, these were, ischaemic heart disease, stroke, chronic obstructive pulmonary disease, and lower respiratory infections. In 2021, however, COVID-19 replaced stroke as the second-leading age-standardised cause of death, with 94·0 deaths (95% UI 89·2-100·0) per 100 000 population. The COVID-19 pandemic shifted the rankings of the leading five causes, lowering stroke to the third-leading and chronic obstructive pulmonary disease to the fourth-leading position. In 2021, the highest age-standardised death rates from COVID-19 occurred in sub-Saharan Africa (271·0 deaths [250·1-290·7] per 100 000 population) and Latin America and the Caribbean (195·4 deaths [182·1-211·4] per 100 000 population). The lowest age-standardised death rates from COVID-19 were in the high-income super-region (48·1 deaths [47·4-48·8] per 100 000 population) and southeast Asia, east Asia, and Oceania (23·2 deaths [16·3-37·2] per 100 000 population). Globally, life expectancy steadily improved between 1990 and 2019 for 18 of the 22 investigated causes. Decomposition of global and regional life expectancy showed the positive effect that reductions in deaths from enteric infections, lower respiratory infections, stroke, and neonatal deaths, among others have contributed to improved survival over the study period. However, a net reduction of 1·6 years occurred in global life expectancy between 2019 and 2021, primarily due to increased death rates from COVID-19 and other pandemic-related mortality. Life expectancy was highly variable between super-regions over the study period, with southeast Asia, east Asia, and Oceania gaining 8·3 years (6·7-9·9) overall, while having the smallest reduction in life expectancy due to COVID-19 (0·4 years). The largest reduction in life expectancy due to COVID-19 occurred in Latin America and the Caribbean (3·6 years). Additionally, 53 of the 288 causes of death were highly concentrated in locations with less than 50% of the global population as of 2021, and these causes of death became progressively more concentrated since 1990, when only 44 causes showed this pattern. The concentration phenomenon is discussed heuristically with respect to enteric and lower respiratory infections, malaria, HIV/AIDS, neonatal disorders, tuberculosis, and measles. INTERPRETATION Long-standing gains in life expectancy and reductions in many of the leading causes of death have been disrupted by the COVID-19 pandemic, the adverse effects of which were spread unevenly among populations. Despite the pandemic, there has been continued progress in combatting several notable causes of death, leading to improved global life expectancy over the study period. Each of the seven GBD super-regions showed an overall improvement from 1990 and 2021, obscuring the negative effect in the years of the pandemic. Additionally, our findings regarding regional variation in causes of death driving increases in life expectancy hold clear policy utility. Analyses of shifting mortality trends reveal that several causes, once widespread globally, are now increasingly concentrated geographically. These changes in mortality concentration, alongside further investigation of changing risks, interventions, and relevant policy, present an important opportunity to deepen our understanding of mortality-reduction strategies. Examining patterns in mortality concentration might reveal areas where successful public health interventions have been implemented. Translating these successes to locations where certain causes of death remain entrenched can inform policies that work to improve life expectancy for people everywhere. FUNDING Bill & Melinda Gates Foundation

Global, regional, and national burden of disorders affecting the nervous system, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

BackgroundDisorders affecting the nervous system are diverse and include neurodevelopmental disorders, late-life neurodegeneration, and newly emergent conditions, such as cognitive impairment following COVID-19. Previous publications from the Global Burden of Disease, Injuries, and Risk Factor Study estimated the burden of 15 neurological conditions in 2015 and 2016, but these analyses did not include neurodevelopmental disorders, as defined by the International Classification of Diseases (ICD)-11, or a subset of cases of congenital, neonatal, and infectious conditions that cause neurological damage. Here, we estimate nervous system health loss caused by 37 unique conditions and their associated risk factors globally, regionally, and nationally from 1990 to 2021.MethodsWe estimated mortality, prevalence, years lived with disability (YLDs), years of life lost (YLLs), and disability-adjusted life-years (DALYs), with corresponding 95% uncertainty intervals (UIs), by age and sex in 204 countries and territories, from 1990 to 2021. We included morbidity and deaths due to neurological conditions, for which health loss is directly due to damage to the CNS or peripheral nervous system. We also isolated neurological health loss from conditions for which nervous system morbidity is a consequence, but not the primary feature, including a subset of congenital conditions (ie, chromosomal anomalies and congenital birth defects), neonatal conditions (ie, jaundice, preterm birth, and sepsis), infectious diseases (ie, COVID-19, cystic echinococcosis, malaria, syphilis, and Zika virus disease), and diabetic neuropathy. By conducting a sequela-level analysis of the health outcomes for these conditions, only cases where nervous system damage occurred were included, and YLDs were recalculated to isolate the non-fatal burden directly attributable to nervous system health loss. A comorbidity correction was used to calculate total prevalence of all conditions that affect the nervous system combined.FindingsGlobally, the 37 conditions affecting the nervous system were collectively ranked as the leading group cause of DALYs in 2021 (443 million, 95% UI 378–521), affecting 3·40 billion (3·20–3·62) individuals (43·1%, 40·5–45·9 of the global population); global DALY counts attributed to these conditions increased by 18·2% (8·7–26·7) between 1990 and 2021. Age-standardised rates of deaths per 100 000 people attributed to these conditions decreased from 1990 to 2021 by 33·6% (27·6–38·8), and age-standardised rates of DALYs attributed to these conditions decreased by 27·0% (21·5–32·4). Age-standardised prevalence was almost stable, with a change of 1·5% (0·7–2·4). The ten conditions with the highest age-standardised DALYs in 2021 were stroke, neonatal encephalopathy, migraine, Alzheimer's disease and other dementias, diabetic neuropathy, meningitis, epilepsy, neurological complications due to preterm birth, autism spectrum disorder, and nervous system cancer.InterpretationAs the leading cause of overall disease burden in the world, with increasing global DALY counts, effective prevention, treatment, and rehabilitation strategies for disorders affecting the nervous system are needed

Influence of physical and biological processes on the seasonal cycle of biogenic flux in the equatorial Indian Ocean

Seasonal cycle of biogenic fluxes obtained from sediment trap at two locations 5°24' N, 86°46' E (southern Bay of Bengal trap; SBBT) and 3°34' N, 77°46' E (equatorial Indian Ocean trap; EIOT) within the equatorial Indian Ocean (EIO) were examined to understand the factors that control them. The sediment trap data at SBBT was collected for ten years from November 1987 while that at EIOT was for a one year period from January 1996. The characteristic of biogenic flux at SBBT was the strong seasonality with peak flux in August, while lack of seasonality characterised the flux at EIOT. The high chlorophyll biomass at the SBBT during the summer monsoon was supported by a combination of processes such as wind-mixing and advection, both of which supplied new nitrogen to the upper ocean. In contrast, the elevated chlorophyll at EIOT during summer monsoon was supported only by wind mixing. High cell counts of phytoplankton (> 5 μm) at SBBT dominated by diatoms suggest the operation of classical food web and high carbon export. On the contrary, dominance of pico-phytoplankton and one-and-a-half time higher magnitude of micro-zooplankton biomass along with 2-fold lesser meso-zooplankton at EIOT indicated the importance of microbial loop. The substantial decrease in the carbon export at EIOT indicated faster remineralization of photosynthetically produced organic matter

Demographic data of thyroiditis from a south Indian city

Background: Thyroiditis involves thyroid gland inflammation due to a wide variety of causes. The common varieties are subacute, silent and postpartum thyroiditis. Aims and Objectives: To retrospectively collect demographic data of thyroiditis from Bangalore over the past 5 years. Materials and Methods: Data were collected from three major nuclear medicine centers in Bangalore of the patients who came for technetium (Tc) 99m pertechnetate scan of the thyroid. The diagnosis was based on the Tc 99 scan evidence of thyroiditis in these patients and biochemical evidence of thyrotoxicosis. Results: The total number of cases recorded were 2513. The females were more commonly affected compared with males with sex distribution of 1698 females and 815 females (2:1). The mean age of females was 32.5 ± 11.3 years whereas the mean age of males was 37.2 ± 12.4 years. The highest numbers of cases were recorded in the months of June and August. Conclusions: The females developed thyroiditis frequently and at an earlier age when compared with males. This data could give us an insight into the demographic pattern of thyroiditis in our country and may help in planning future preventive strategies

Scintillation Proximity Assay for Inhibitors of Escherichia coli MurG and, Optionally, MraY

MurG and MraY, essential enzymes involved in the synthesis of bacterial peptidoglycan, are difficult to assay because the substrates are lipidic and hard to prepare in large quantities. Based on the use of Escherichia coli membranes lacking PBP1b, we report a high-throughput method to measure the activity of MurG and, optionally, MraY as well. In these membranes, incubation with the two peptidoglycan sugar precursors results in accumulation of lipid II rather than the peptidoglycan produced by wild-type membranes. MurG was assayed by addition of UDP-[(3)H]N-acetylglucosamine to membranes in which lipid I was preformed by incubation with UDP-N-acetyl-muramylpentapeptide, and the product was captured by wheat germ agglutinin scintillation proximity assay beads. In a modification of the assay, the activity of MraY was coupled to that of MurG by addition of both sugar precursors together in a single step. This allows simultaneous detection of inhibitors of either enzyme. Both assays could be performed using wild-type membranes by addition of the transglycosylase inhibitor moenomycin. Nisin and vancomycin inhibited the MurG reaction; the MraY-MurG assay was inhibited by tunicamycin as well. Inhibitors of other enzymes of peptidoglycan synthesis—penicillin G, moenomycin, and bacitracin—had no effect. Surprisingly, however, the β-lactam cephalosporin C inhibited both the MurG and MraY-MurG assays, indicating a secondary mechanism by which this drug inhibits bacterial growth. In addition, it inhibited NADH dehydrogenase in membranes, a hitherto-unreported activity. These assays can be used to screen for novel antibacterial agents