School-based interventions modestly increase physical activity and cardiorespiratory fitness but are least effective for youth who need them most: an individual participant pooled analysis of 20 controlled trials.

OBJECTIVES: To determine if subpopulations of students benefit equally from school-based physical activity interventions in terms of cardiorespiratory fitness and physical activity. To examine if physical activity intensity mediates improvements in cardiorespiratory fitness. DESIGN: Pooled analysis of individual participant data from controlled trials that assessed the impact of school-based physical activity interventions on cardiorespiratory fitness and device-measured physical activity. PARTICIPANTS: Data for 6621 children and adolescents aged 4-18 years from 20 trials were included. MAIN OUTCOME MEASURES: Peak oxygen consumption (VO2Peak mL/kg/min) and minutes of moderate and vigorous physical activity. RESULTS: Interventions modestly improved students' cardiorespiratory fitness by 0.47 mL/kg/min (95% CI 0.33 to 0.61), but the effects were not distributed equally across subpopulations. Girls and older students benefited less than boys and younger students, respectively. Students with lower levels of initial fitness, and those with higher levels of baseline physical activity benefitted more than those who were initially fitter and less active, respectively. Interventions had a modest positive effect on physical activity with approximately one additional minute per day of both moderate and vigorous physical activity. Changes in vigorous, but not moderate intensity, physical activity explained a small amount (~5%) of the intervention effect on cardiorespiratory fitness. CONCLUSIONS: Future interventions should include targeted strategies to address the needs of girls and older students. Interventions may also be improved by promoting more vigorous intensity physical activity. Interventions could mitigate declining youth cardiorespiratory fitness, increase physical activity and promote cardiovascular health if they can be delivered equitably and their effects sustained at the population level.

The Critical E4 State of Nitrogenase Catalysis.

The reaction catalyzed by the nitrogenase enzyme involves breaking the stable triple bond of the dinitrogen molecule and is consequently considered among the most challenging reactions in biology. While many aspects regarding its atomic mechanism remain to be elucidated, a kinetic scheme established by David Lowe and Roger Thorneley has remained a gold standard for functional studies of the enzyme for more than 30 years. Recent three-dimensional structures of ligand-bound states of molybdenum- and vanadium-dependent nitrogenases have revealed the actual site of substrate binding on the large active site cofactors of this class of enzymes. The binding mode of an inhibitor and a reaction intermediate further substantiate a hypothesis by Seefeldt, Hoffman, and Dean that the activation of N2 is made possible by a reductive elimination of H2 that leaves the cofactor in a super-reduced state that can bind and reduce the inert N2 molecule. Here we discuss the immediate implications of the structurally observed mode of binding of small molecules to the enzyme with respect to the early stages of the Thorneley-Lowe mechanism of nitrogenase. Four consecutive single-electron reductions give rise to two bridging hydrides at the cluster surface that can recombine to eliminate H2 and enable the reduced cluster to bind its substrate in a bridging mode.

The flavinyl transferase ApbE of Pseudomonas stutzeri matures the NosR protein required for nitrous oxide reduction.

The copper-containing enzyme nitrous oxide reductase (N2OR) catalyzes the transformation of nitrous oxide (N2O) to dinitrogen (N2) in microbial denitrification. Several accessory factors are essential for assembling the two copper sites CuA and CuZ, and for maintaining the activity. In particular, the deletion of either the transmembrane iron-sulfur flavoprotein NosR or the periplasmic protein NosX, a member of the ApbE family, abolishes N2O respiration. Here we demonstrate through biochemical and structural studies that the ApbE protein from Pseudomonas stutzeri, where the nosX gene is absent, is a monomeric FAD-binding protein that can serve as the flavin donor for NosR maturation via covalent flavinylation of a threonine residue. The flavin transfer reaction proceeds both in vivo and in vitro to generate post-translationally modified NosR with covalently bound FMN. Only FAD can act as substrate and the reaction requires a divalent cation, preferably Mg2+ that was also present in the crystal structure. In addition, the reaction is species-specific to a certain extent.

Production and isolation of vanadium nitrogenase from Azotobacter vinelandii by molybdenum depletion.

The alternative, vanadium-dependent nitrogenase is employed by Azotobacter vinelandii for the fixation of atmospheric N2 under conditions of molybdenum starvation. While overall similar in architecture and functionality to the common Mo-nitrogenase, the V-dependent enzyme exhibits a series of unique features that on one hand are of high interest for biotechnological applications. As its catalytic properties differ from Mo-nitrogenase, it may on the other hand also provide invaluable clues regarding the molecular mechanism of biological nitrogen fixation that remains scarcely understood to date. Earlier studies on vanadium nitrogenase were almost exclusively based on a ΔnifHDK strain of A. vinelandii, later also in a version with a hexahistidine affinity tag on the enzyme. As structural analyses remained unsuccessful with such preparations we have developed protocols to isolate unmodified vanadium nitrogenase from molybdenum-depleted, actively nitrogen-fixing A. vinelandii wild-type cells. The procedure provides pure protein at high yields whose spectroscopic properties strongly resemble data presented earlier. Analytical size-exclusion chromatography shows this preparation to be a VnfD2K2G2 heterohexamer.

A school-based intervention improved dietary intake outcomes and reduced waist circumference in adolescents: a cluster randomized controlled trial.

BACKGROUND: In Ecuador, adolescents' food intake does not comply with guidelines for a healthy diet. Together with abdominal obesity adolescent's inadequate diets are risk factors for non-communicable diseases. We report the effectiveness of a school-based intervention on the dietary intake and waist circumference among Ecuadorian adolescents. METHODS: A pair-matched cluster randomized controlled trial including 1430 adolescents (12-14 years old) was conducted. The program aimed at improving the nutritional value of dietary intake, physical activity (primary outcomes), body mass index, waist circumference and blood pressure (secondary outcomes). This paper reports: (i) the effect on fruit and vegetable intake, added sugar intake, unhealthy snacking (consumption of unhealthy food items that are not in line with the dietary guidelines eaten during snack time; i.e. table sugar, sweets, salty snacks, fast food, soft drinks and packaged food), breakfast intake and waist circumference; and, (ii) dose and reach of the intervention. Dietary outcomes were estimated by means of two 24-h recall at baseline, after the first 17-months (stage one) and after the last 11-months (stage two) of implementation. Dose and reach were evaluated using field notes and attendance forms. Educational toolkits and healthy eating workshops with parents and food kiosks staff in the schools were implemented in two different stages. The overall effect was assessed using linear mixed models and regression spline mixed effect models were applied to evaluate the effect after each stage. RESULTS: Data from 1046 adolescents in 20 schools were analyzed. Participants from the intervention group consumed lower quantities of unhealthy snacks (-23.32 g; 95% CI: -45.25,-1.37) and less added sugar (-5.66 g; 95% CI: -9.63,-1.65) at the end of the trial. Daily fruit and vegetable intake decreased in both the intervention and control groups compared to baseline, albeit this decrease was 23.88 g (95% CI: 7.36, 40.40) lower in the intervention group. Waist circumference (-0.84 cm; 95% CI: -1.68, 0.28) was lower in the intervention group at the end of the program; the effect was mainly observed at stage one. Dose and reach were also higher at stage one. CONCLUSIONS: The trial had positive effects on risk factors for non-communicable diseases, i.e. decreased consumption of unhealthy snacks. The program strategies must be implemented at the national level through collaboration between the academia and policy makers to assure impact at larger scale. TRIAL REGISTRATION: ClinicalTrial.gov-NCT01004367 .

Direct observation of electrogenic NH4(+) transport in ammonium transport (Amt) proteins.

Ammonium transport (Amt) proteins form a ubiquitous family of integral membrane proteins that specifically shuttle ammonium across membranes. In prokaryotes, archaea, and plants, Amts are used as environmental NH4(+) scavengers for uptake and assimilation of nitrogen. In the eukaryotic homologs, the Rhesus proteins, NH4(+)/NH3 transport is used instead in acid-base and pH homeostasis in kidney or NH4(+)/NH3 (and eventually CO2) detoxification in erythrocytes. Crystal structures and variant proteins are available, but the inherent challenges associated with the unambiguous identification of substrate and monitoring of transport events severely inhibit further progress in the field. Here we report a reliable in vitro assay that allows us to quantify the electrogenic capacity of Amt proteins. Using solid-supported membrane (SSM)-based electrophysiology, we have investigated the three Amt orthologs from the euryarchaeon Archaeoglobus fulgidus. Af-Amt1 and Af-Amt3 are electrogenic and transport the ammonium and methylammonium cation with high specificity. Transport is pH-dependent, with a steep decline at pH values of ∼5.0. Despite significant sequence homologies, functional differences between the three proteins became apparent. SSM electrophysiology provides a long-sought-after functional assay for the ubiquitous ammonium transporters.

Two years of school-based intervention program could improve the physical fitness among Ecuadorian adolescents at health risk: subgroups analysis from a cluster-randomized trial.

BACKGROUND: Adolescents with overweight and poor physical fitness have an increased likelihood of developing cardiovascular diseases during adulthood. In Ecuador, a health promotion program improved the muscular strength and speed-agility, and reduced the decline of the moderate-to-vigorous physical activity of adolescents after 28 months. We performed a sub-group analysis to assess the differential effect of this intervention in overweight and low-fit adolescents. METHODS: We performed a cluster-randomized pair matched trial in schools located in Cuenca-Ecuador. In total 20 schools (clusters) were pair matched, and 1440 adolescents of grade 8 and 9 (mean age of 12.3 and 13.3 years respectively) participated in the trial. For the purposes of the subgroup analysis, the adolescents were classified into groups according to their weight status (body mass index) and aerobic capacity (scores in the 20 m shuttle run and FITNESSGRAM standards) at baseline. Primary outcomes included physical fitness (vertical jump, speed shuttle run) and physical activity (proportion of students achieving over 60 min of moderate-to-vigorous physical activity/day). For these primary outcomes, we stratified analysis by weight (underweight, normal BMI and overweight/obese) and fitness (fit and low fitness) groups. Mixed linear regression models were used to assess the intervention effect. RESULTS: The prevalence of overweight/obesity, underweight and poor physical fitness was 20.3 %, 5.8 % and 84.8 % respectively. A higher intervention effect was observed for speed shuttle run in overweight (ß = -1.85 s, P = 0.04) adolescents compared to underweight (ß = -1.66 s, P = 0.5) or normal weight (ß = -0.35 s, P = 0.6) peers. The intervention effect on vertical jump was higher in adolescents with poor physical fitness (ß = 3.71 cm, P = 0.005) compared to their fit peers (ß = 1.28 cm, P = 0.4). The proportion of students achieving over 60 min of moderate-to-vigorous physical activity/day was not significantly different according to weight or fitness status. CONCLUSION: Comprehensive school-based interventions that aim to improve diet and physical activity could improve speed and strength aspects of physical fitness in low-fit and overweight/obese adolescents. TRIAL REGISTRATION: Clinicaltrials.gov identifier NCT01004367 . Registered October 28, 2009.

School-based intervention on healthy behaviour among Ecuadorian adolescents: effect of a cluster-randomized controlled trial on screen-time.

BACKGROUND: Effective interventions on screen-time behaviours (television, video games and computer time) are needed to prevent non-communicable diseases in low- and middle-income countries. The present manuscript investigates the effect of a school-based health promotion intervention on screen-time behaviour among 12- to 15-year-old adolescents. We report the effect of the trial on screen-time after two stages of implementation. METHODS: We performed a cluster-randomised pair matched trial in urban schools in Cuenca-Ecuador. Participants were adolescents of grade eight and nine (mean age 12.8 ± 0.8 years, n = 1370, control group n = 684) from 20 schools (control group n = 10). The intervention included an individual and environmental component tailored to the local context and resources. The first intervention stage focused on diet, physical activity and screen-time behaviour, while the second stage focused only on diet and physical activity. Screen-time behaviours, primary outcome, were assessed at baseline, after the first (18 months) and second stage (28 months). Mixed linear models were used to analyse the data. RESULTS: After the first stage (data from n = 1224 adolescents; control group n = 608), the intervention group had a lower increase in TV-time on a week day (ß = -15.7 min; P = 0.003) and weekend day (ß = -18.9 min; P = 0.005), in total screen-time on a weekday (ß = -25.9 min; P = 0.03) and in the proportion of adolescents that did not meet the screen-time recommendation (ß = -4 percentage point; P = 0.01), compared to the control group. After the second stage (data from n = 1078 adolescents; control group n = 531), the TV-time on a weekday (ß = 13.1 min; P = 0.02), and total screen-time on a weekday (ß = 21.4 min; P = 0.03) increased more in adolescents from the intervention group. No adverse effects were reported. DISCUSSION AND CONCLUSION: A multicomponent school-based intervention was only able to mitigate the increase in adolescents' television time and total screen-time after the first stage of the intervention or in other words, when the intervention included specific components or activities that focused on reducing screen-time. After the second stage of the intervention, which only included components and activities related to improve healthy diet and physical activity and not to decrease the screen-time, the adolescents increased their screen-time again. Our findings might imply that reducing screen-time is only possible when the intervention focuses specifically on reducing screen-time. TRIAL REGISTRATION: Clinicaltrials.gov identifier NCT01004367.

Structural and functional characterization of the nitrite channel NirC from Salmonella typhimurium.

Nitrite (NO(2)(-)) is a central intermediate in the nitrogen metabolism of microorganisms and plants, and is used as a cytotoxin by macrophages as part of the innate immune response. The bacterial membrane protein NirC acts as a specific channel to facilitate the transport of nitrite anions across lipid bilayers for cytoplasmic detoxification. Despite NirC's importance in nitrogen metabolism and in the pathogenicity of enteric bacteria, available biochemical data are scarce. Here we present a functional and structural characterization of NirC from Salmonella typhimurium by lipid bilayer electrophysiology and X-ray crystallography. NirC is a pentameric member of the formate/nitrite transporter family of membrane proteins that operates as a channel with high conductance. Single-channel measurements reveal fast and slow gating events but, in contrast to the related FocA formate channel, no pH-dependent gating. A 2.4Å crystal structure of NirC at pH 5 shows similarity to FocA and aquaporins, but lacks the structural asymmetry observed in the formate channel at similarly low pH. Resolved water molecules in the protomers suggest a transport mechanism that also permits a facultative NO(2)(-)/H(+) symport.

The formate channel FocA exports the products of mixed-acid fermentation.

Formate is a major metabolite in the anaerobic fermentation of glucose by many enterobacteria. It is translocated across cellular membranes by the pentameric ion channel/transporter FocA that, together with the nitrite channel NirC, forms the formate/nitrite transporter (FNT) family of membrane transport proteins. Here we have carried out an electrophysiological analysis of FocA from Salmonella typhimurium to characterize the channel properties and assess its specificity toward formate and other possible permeating ions. Single-channel currents for formate, hypophosphite and nitrite revealed two mechanistically distinct modes of gating that reflect different types of structural rearrangements in the transport channel of each FocA protomer. Moreover, FocA did not conduct cations or divalent anions, but the chloride anion was identified as further transported species, along with acetate, lactate and pyruvate. Formate, acetate and lactate are major end products of anaerobic mixed-acid fermentation, the pathway where FocA is predominantly required, so that this channel is ideally adapted to act as a multifunctional export protein to prevent their intracellular accumulation. Because of the high degree of conservation in the residues forming the transport channel among FNT family members, the flexibility in conducting multiple molecules is most likely a general feature of these proteins.

Insights into the bioactivity of mensacarcin and epoxide formation by MsnO8.

Mensacarcin, a potential antitumour drug, is produced by Streptomyces bottropensis. The structure consists of a three-membered ring system with many oxygen atoms. Of vital importance in this context is an epoxy moiety in the side chain of mensacarcin. Our studies with different mensacarcin derivatives have demonstrated that this epoxy group is primarily responsible for the cytotoxic effect of mensacarcin. In order to obtain further information about this epoxy moiety, inactivation experiments in the gene cluster were carried out to identify the epoxy-forming enzyme. Therefore the cosmid cos2, which covers almost the complete type II polyketide synthase (PKS) gene cluster, was heterologously expressed in Streptomyces albus. This led to production of didesmethylmensacarcin, due to the fact that methyltransferase genes are missing in the cosmid. Further gene inactivation experiments on this cosmid showed that MsnO8, a luciferase-like monooxygenase, introduces the epoxy group at the end of the biosynthesis of mensacarcin. In addition, the protein MsnO8 was purified, and its crystal structure was determined to a resolution of 1.80 Å.

Catalytic scope of the thiamine-dependent multifunctional enzyme cyclohexane-1,2-dione hydrolase.

The thiamine diphosphate (ThDP)-dependent enzyme cyclohexane-1,2-dione hydrolase (CDH) was expressed in Escherichia coli and purified by affinity chromatography (Ni-NTA). Recombinant CDH showed the same C-C bond-cleavage and C-C bond-formation activities as the native enzyme. Furthermore, we have shown that CDH catalyzes the asymmetric cross-benzoin reaction of aromatic aldehydes and (decarboxylated) pyruvate (up to quantitative conversion, 92-99 % ee). CDH accepts also hydroxybenzaldehydes and nitrobenzaldehydes; these previously have not (or only in rare cases) been known as substrates of other ThDP-dependent enzymes. On a semipreparative scale, sterically demanding 4-(tert-butyl)benzaldehyde and 2-naphthaldehyde were transformed into the corresponding 2-hydroxy ketone products in high yields. Additionally, certain benzaldehydes with electron withdrawing substituents were identified as potential inhibitors of the ligase activity of CDH.

A school-based intervention improves physical fitness in Ecuadorian adolescents: a cluster-randomized controlled trial.

BACKGROUND: Effective lifestyle interventions are needed to prevent noncommunicable diseases in low- and middle-income countries. We analyzed the effects of a school-based health promotion intervention on physical fitness after 28 months and explored if the effect varied with important school characteristics. We also assessed effects on screen time, physical activity and BMI. METHODS AND RESULTS: We performed a cluster-randomized pair matched trial in schools in urban Ecuador. The intervention included an individual and environmental component tailored to the local context and resources. Primary outcomes were physical fitness (EUROFIT battery), screen time (questionnaires) and physical activity (accelerometers). Change in BMI was a secondary outcome. A total of 1440 grade 8 and 9 adolescents (intervention: n = 700, 48.6%) and 20 schools (intervention: n = 10, 50%) participated. Data of 1083 adolescents (intervention: n = 550, 50.8%) from 20 schools were analyzed. CONCLUSIONS: A school-based intervention with an individual and environment component can improve physical fitness and can minimize the decline in physical activity levels from childhood into adolescence in urban Ecuador. TRIAL REGISTRATION: Clinicaltrials.gov identifier NCT01004367.

Asymmetric Stetter reactions catalyzed by thiamine diphosphate-dependent enzymes.

The intermolecular asymmetric Stetter reaction is an almost unexplored transformation for biocatalysts. Previously reported thiamine diphosphate (ThDP)-dependent PigD from Serratia marcescens is the first enzyme identified to catalyze the Stetter reaction of α,ß-unsaturated ketones (Michael acceptor substrates) and α-keto acids. PigD is involved in the biosynthesis of the potent cytotoxic agent prodigiosin. Here, we describe the investigation of two new ThDP-dependent enzymes, SeAAS from Saccharopolyspora erythraea and HapD from Hahella chejuensis. Both show a high degree of homology to the amino acid sequence of PigD (39 and 51 %, respectively). The new enzymes were heterologously overproduced in Escherichia coli, and the yield of soluble protein was enhanced by co-expression of the chaperone genes groEL/ES. SeAAS and HapD catalyze intermolecular Stetter reactions in vitro with high enantioselectivity. The enzymes possess a characteristic substrate range with respect to Michael acceptor substrates. This provides support for a new type of ThDP-dependent enzymatic activity, which is abundant in various species and not restricted to prodigiosin biosynthesis in different strains. Moreover, PigD, SeAAS, and HapD are also able to catalyze asymmetric carbon-carbon bond formation reactions of aldehydes and α-keto acids, resulting in 2-hydroxy ketones.

Dietary intake practices associated with cardiovascular risk in urban and rural Ecuadorian adolescents: a cross-sectional study.

BACKGROUND: Cardiovascular diseases (CVD) are amongst the leading causes of death worldwide. Risk factors of CVD develop during childhood and adolescence, and dietary quality has been linked to the development of CVD itself. This study examines the association between dietary patterns and cardiovascular risk in a group of urban and rural Ecuadorian adolescents from different socioeconomic backgrounds. METHODS: A cross-sectional study was conducted from January 2008 to April 2009 among 606 adolescents from the 8th, 9th and 10th grade in an urban area (Cuenca), and 173 adolescents from a rural area (Nabón) in Ecuador. Data collection involved measuring anthropometric data (weight, height and waist circumference), blood pressure, dietary intake (2-day 24 h recall) and socio-demographic characteristics. Fasting blood lipids and glucose were measured in a subsample of 334 adolescents. Factor analysis was used to identify dietary patterns and linear regression models were used to (i) identify differences in food intake practices according to socioeconomic status and place of residence and (ii) establish relationships between dietary patterns and cardiovascular risk factors. RESULTS: Median energy intake was 1851 kcal/day. Overall, fiber, fish and fruit and vegetables were scarcely consumed, while added sugar, refined cereals and processed food were important constituents of the diet. Two dietary patterns emerged, one labelled as "rice-rich non-animal fat pattern" and the other one as "wheat-dense animal-fat pattern". The first pattern was correlated with a moderate increase in glucose in urban participants, while the second pattern was associated with higher LDL and cholesterol blood levels in rural participants. CONCLUSIONS: This group of adolescents presented various dietary practices conducive to CVD development. Effective strategies are needed to prevent CVD in the Ecuadorian population by encouraging a balanced diet, which contains less refined cereals, added sugar, and processed food, but has more fruits, vegetables and whole grain cereals.

Physical fitness among urban and rural Ecuadorian adolescents and its association with blood lipids: a cross sectional study.

BACKGROUND: Physical fitness has been proposed as a marker for health during adolescence. Currently, little is known about physical fitness and its association with blood lipid profile in adolescents from low and middle-income countries. The aim of this study is therefore to assess physical fitness among urban and rural adolescents and its associations with blood lipid profile in a middle-income country. METHODS: A cross-sectional study was conducted between January 2008 and April 2009 in 648 Ecuadorian adolescents (52.3% boys), aged 11 to 15 years, attending secondary schools in Cuenca (urban n = 490) and Nabón (rural n = 158). Data collection included anthropometric measures, application of the EUROFIT battery, dietary intake (2-day 24 h recall), socio-demographic characteristics, and blood samples from a subsample (n = 301). The FITNESGRAM standards were used to evaluate fitness. The associations of fitness and residential location with blood lipid profile were assessed by linear and logistic regression after adjusting for confounding factors. RESULTS: The majority (59%) of the adolescents exhibited low levels of aerobic capacity as defined by the FITNESSGRAM standards. Urban adolescents had significantly higher mean scores in five EUROFIT tests (20 m shuttle, speed shuttle run, plate tapping, sit-up and vertical jump) and significantly most favorable improved plasma lipid profile (triglycerides and HDL) as compared to rural adolescents. There was a weak association between blood lipid profile and physical fitness in both urban and rural adolescents, even after adjustment for confounding factors. CONCLUSIONS: Physical fitness, in our sample of Ecuadorian adolescents, was generally poor. Urban adolescents had better physical fitness and blood lipid profiles than rural adolescents. The differences in fitness did not explain those in blood lipid profile between urban and rural adolescents.

Extended reaction scope of thiamine diphosphate dependent cyclohexane-1,2-dione hydrolase: from C-C bond cleavage to C-C bond ligation.

ThDP-dependent cyclohexane-1,2-dione hydrolase (CDH) catalyzes the CC bond cleavage of cyclohexane-1,2-dione to 6-oxohexanoate, and the asymmetric benzoin condensation between benzaldehyde and pyruvate. One of the two reactivities of CDH was selectively knocked down by mutation experiments. CDH-H28A is much less able to catalyze the CC bond formation, while the ability for CC bond cleavage is still intact. The double variant CDH-H28A/N484A shows the opposite behavior and catalyzes the addition of pyruvate to cyclohexane-1,2-dione, resulting in the formation of a tertiary alcohol. Several acyloins of tertiary alcohols are formed with 54-94 % enantiomeric excess. In addition to pyruvate, methyl pyruvate and butane-2,3-dione are alternative donor substrates for CC bond formation. Thus, the very rare aldehyde-ketone cross-benzoin reaction has been solved by design of an enzyme variant.

How sensor Amt-like proteins integrate ammonium signals.

Unlike aquaporins or potassium channels, ammonium transporters (Amts) uniquely discriminate ammonium from potassium and water. This feature has certainly contributed to their repurposing as ammonium receptors during evolution. Here, we describe the ammonium receptor Sd-Amt1, where an Amt module connects to a cytoplasmic diguanylate cyclase transducer module via an HAMP domain. Structures of the protein with and without bound ammonium were determined to 1.7- and 1.9-Ångstrom resolution, depicting the ON and OFF states of the receptor and confirming the presence of a binding site for two ammonium cations that is pivotal for signal perception and receptor activation. The transducer domain was disordered in the crystals, and an AlphaFold2 prediction suggests that the helices linking both domains are flexible. While the sensor domain retains the trimeric fold formed by all Amt family members, the HAMP domains interact as pairs and serve to dimerize the transducer domain upon activation.

The formate/nitrite transporter family of anion channels.

The formate/nitrite transporter (FNT) family of integral membrane proteins comprises pentameric channels for monovalent anions that exhibit a broad specificity for small anions such as chloride, the physiological cargo molecules formate, nitrite, and hydrosulfide, and also larger organic acids. Three-dimensional structures are available for the three known subtypes, FocA, NirC, and HSC, which reveal remarkable evolutionary optimizations for the respective physiological context of the channels. FNT channels share a conserved translocation pathway in each protomer, with a central hydrophobic cavity that is separated from both sides of the membrane by a narrow constriction. A single protonable residue, a histidine, plays a key role by transiently protonating the transported anion to allow an uncharged species to pass the hydrophobic barrier. Further selectivity is reached through variations in the electrostatic surface potential of the proteins, priming the formate channel FocA for anion export, whereas NirC and HSC should work bidirectionally. Electrophysiological studies have shown that a broad variety of monovalent anions can be transported, and in the case of FocA, these match exactly the products of mixed-acid fermentation, the predominant metabolic pathway for most enterobacterial species.

α-Hydroxy-ß-keto acid rearrangement-decarboxylation: impact on thiamine diphosphate-dependent enzymatic transformations.

The thiamine diphosphate (ThDP) dependent MenD catalyzes the reaction of α-ketoglutarate with pyruvate to selectively form 4-hydroxy-5-oxohexanoic acid 2, which seems to be inconsistent with the assumed acyl donor role of the physiological substrate α-KG. In contrast the reaction of α-ketoglutarate with acetaldehyde gives exclusively the expected 5-hydroxy-4-oxo regioisomer 1. These reactions were studied by NMR and CD spectroscopy, which revealed that with pyruvate the observed regioselectivity is due to the rearrangement-decarboxylation of the initially formed α-hydroxy-ß-keto acid rather than a donor-acceptor substrate role variation. Further experiments with other ThDP-dependent enzymes, YerE, SucA, and CDH, verified that this degenerate decarboxylation can be linked to the reduced enantioselectivity of acyloins often observed in ThDP-dependent enzymatic transformations.