The crystal structure of Shethna protein II (FeSII) from Azotobacter vinelandii suggests a domain swap.

The Azotobacter vinelandii FeSII protein forms an oxygen-resistant complex with the nitrogenase MoFe and Fe proteins. FeSII is an adrenodoxin-type ferredoxin that forms a dimer in solution. Previously, the crystal structure was solved [Schlesier et al. (2016), J. Am. Chem. Soc. 138, 239-247] with five copies in the asymmetric unit. One copy is a normal adrenodoxin domain that forms a dimer with its crystallographic symmetry mate. The other four copies are in an `open' conformation with a loop flipped out exposing the 2Fe-2S cluster. The open and closed conformations were interpreted as oxidized and reduced, respectively, and the large conformational change in the open configuration allowed binding to nitrogenase. Here, the structure of FeSII was independently solved in the same crystal form. The positioning of the atoms in the unit cell is similar to the earlier report. However, the interpretation of the structure is different. The `open' conformation is interpreted as the product of a crystallization-induced domain swap. The 2Fe-2S cluster is not exposed to solvent, but in the crystal its interacting helix is replaced by the same helix residues from a crystal symmetry mate. The domain swap is complicated, as it is unusual in being in the middle of the protein rather than at a terminus, and it creates arrangements of molecules that can be interpreted in multiple ways. It is also cautioned that crystal structures should be interpreted in terms of the contents of the entire crystal rather than of one asymmetric unit.

Dynamic lid domain of Chloroflexus aurantiacus Malonyl-CoA reductase controls the reaction.

Malonyl-Coenzyme A Reductase (MCR) in Chloroflexus aurantiacus, a characteristic enzyme of the 3-hydroxypropionate (3-HP) cycle, catalyses the reduction of malonyl-CoA to 3-HP. MCR is a bi-functional enzyme; in the first step, malonyl-CoA is reduced to the free intermediate malonate semialdehyde by the C-terminal region of MCR, and this is further reduced to 3-HP by the N-terminal region of MCR. Here we present the crystal structures of both N-terminal and C-terminal regions of the MCR from C. aurantiacus. A catalytic mechanism is suggested by ligand and substrate bound structures, and structural and kinetic studies of MCR variants. Both MCR structures reveal one catalytic, and one non-catalytic SDR (short chain dehydrogenase/reductase) domain. C-terminal MCR has a lid domain which undergoes a conformational change and controls the reaction. In the proposed mechanism of the C-terminal MCR, the conversion of malonyl-CoA to malonate semialdehyde is based on the reduction of malonyl-CoA by NADPH, followed by the decomposition of the hemithioacetal to produce malonate semialdehyde and coenzyme A. Conserved arginines, Arg734 and Arg773 are proposed to play key roles in the mechanism and conserved Ser719, and Tyr737 are other essential residues forming an oxyanion hole for the substrate intermediates.

Toward a glycyl radical enzyme containing synthetic bacterial microcompartment to produce pyruvate from formate and acetate.

Formate has great potential to function as a feedstock for biorefineries because it can be sustainably produced by a variety of processes that don't compete with agricultural production. However, naturally formatotrophic organisms are unsuitable for large-scale cultivation, difficult to engineer, or have inefficient native formate assimilation pathways. Thus, metabolic engineering needs to be developed for model industrial organisms to enable efficient formatotrophic growth. Here, we build a prototype synthetic formate utilizing bacterial microcompartment (sFUT) encapsulating the oxygen-sensitive glycyl radical enzyme pyruvate formate lyase and a phosphate acyltransferase to convert formate and acetyl-phosphate into the central biosynthetic intermediate pyruvate. This metabolic module offers a defined environment with a private cofactor coenzyme A that can cycle efficiently between the encapsulated enzymes. To facilitate initial design-build-test-refine cycles to construct an active metabolic core, we used a "wiffleball" architecture, defined as an icosahedral bacterial microcompartment (BMC) shell with unoccupied pentameric vertices to freely permit substrate and product exchange. The resulting sFUT prototype wiffleball is an active multi enzyme synthetic BMC functioning as platform technology.

Crystal structure of the [2Fe-2S] protein I (Shethna protein I) from Azotobacter vinelandii.

Azotobacter vinelandii is a model diazotroph and is the source of most nitrogenase material for structural and biochemical work. Azotobacter can grow in above-atmospheric levels of oxygen, despite the sensitivity of nitrogenase activity to oxygen. Azotobacter has many iron-sulfur proteins in its genome, which were identified as far back as the 1960s and probably play roles in the complex redox chemistry that Azotobacter must maintain when fixing nitrogen. Here, the 2.1 Šresolution crystal structure of the [2Fe-2S] protein I (Shethna protein I) from A. vinelandii is presented, revealing a homodimer with the [2Fe-2S] cluster coordinated by the surrounding conserved cysteine residues. It is similar to the structure of the thioredoxin-like [2Fe-2S] protein from Aquifex aeolicus, including the positions of the [2Fe-2S] clusters and conserved cysteine residues. The structure of Shethna protein I will provide information for understanding its function in relation to nitrogen fixation and its evolutionary relationships to other ferredoxins.

Replacing the Calvin cycle with the reductive glycine pathway in Cupriavidus necator.

Formate can be directly produced from CO2 and renewable electricity, making it a promising microbial feedstock for sustainable bioproduction. Cupriavidus necator is one of the few biotechnologically-relevant hosts that can grow on formate, but it uses the Calvin cycle, the high ATP cost of which limits biomass and product yields. Here, we redesign C. necator metabolism for formate assimilation via the synthetic, highly ATP-efficient reductive glycine pathway. First, we demonstrate that the upper pathway segment supports glycine biosynthesis from formate. Next, we explore the endogenous route for glycine assimilation and discover a wasteful oxidation-dependent pathway. By integrating glycine biosynthesis and assimilation we are able to replace C. necator's Calvin cycle with the synthetic pathway and achieve formatotrophic growth. We then engineer more efficient glycine metabolism and use short-term evolution to optimize pathway activity. The final growth yield we achieve (2.6 gCDW/mole-formate) nearly matches that of the WT strain using the Calvin Cycle (2.9 gCDW/mole-formate). We expect that further rational and evolutionary optimization will result in a superior formatotrophic C. necator strain, paving the way towards realizing the formate bio-economy.

Structural basis of light-induced redox regulation in the Calvin-Benson cycle in cyanobacteria.

Plants, algae, and cyanobacteria fix carbon dioxide to organic carbon with the Calvin-Benson (CB) cycle. Phosphoribulokinase (PRK) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are essential CB-cycle enzymes that control substrate availability for the carboxylation enzyme Rubisco. PRK consumes ATP to produce the Rubisco substrate ribulose bisphosphate (RuBP). GAPDH catalyzes the reduction step of the CB cycle with NADPH to produce the sugar glyceraldehyde 3-phosphate (GAP), which is used for regeneration of RuBP and is the main exit point of the cycle. GAPDH and PRK are coregulated by the redox state of a conditionally disordered protein CP12, which forms a ternary complex with both enzymes. However, the structural basis of CB-cycle regulation by CP12 is unknown. Here, we show how CP12 modulates the activity of both GAPDH and PRK. Using thermophilic cyanobacterial homologs, we solve crystal structures of GAPDH with different cofactors and CP12 bound, and the ternary GAPDH-CP12-PRK complex by electron cryo-microscopy, we reveal that formation of the N-terminal disulfide preorders CP12 prior to binding the PRK active site, which is resolved in complex with CP12. We find that CP12 binding to GAPDH influences substrate accessibility of all GAPDH active sites in the binary and ternary inhibited complexes. Our structural and biochemical data explain how CP12 integrates responses from both redox state and nicotinamide dinucleotide availability to regulate carbon fixation.

A low-potential terminal oxidase associated with the iron-only nitrogenase from the nitrogen-fixing bacterium Azotobacter vinelandii.

The biological route for nitrogen gas entering the biosphere is reduction to ammonia by the nitrogenase enzyme, which is inactivated by oxygen. Three types of nitrogenase exist, the least-studied of which is the iron-only nitrogenase. The Anf3 protein in the bacterium Rhodobacter capsulatus is essential for diazotrophic (i.e. nitrogen-fixing) growth with the iron-only nitrogenase, but its enzymatic activity and function are unknown. Here, we biochemically and structurally characterize Anf3 from the model diazotrophic bacterium Azotobacter vinelandii Determining the Anf3 crystal structure to atomic resolution, we observed that it is a dimeric flavocytochrome with an unusually close interaction between the heme and the FAD cofactors. Measuring the reduction potentials by spectroelectrochemical redox titration, we observed values of -420 ± 10 and -330 ± 10 mV for the two FAD potentials and -340 ± 1 mV for the heme. We further show that Anf3 accepts electrons from spinach ferredoxin and that Anf3 consumes oxygen without generating superoxide or hydrogen peroxide. We predict that Anf3 protects the iron-only nitrogenase from oxygen inactivation by functioning as an oxidase in respiratory protection, with flavodoxin or ferredoxin as the physiological electron donors.

Design and in vitro realization of carbon-conserving photorespiration.

Photorespiration recycles ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) oxygenation product, 2-phosphoglycolate, back into the Calvin Cycle. Natural photorespiration, however, limits agricultural productivity by dissipating energy and releasing CO2 Several photorespiration bypasses have been previously suggested but were limited to existing enzymes and pathways that release CO2 Here, we harness the power of enzyme and metabolic engineering to establish synthetic routes that bypass photorespiration without CO2 release. By defining specific reaction rules, we systematically identified promising routes that assimilate 2-phosphoglycolate into the Calvin Cycle without carbon loss. We further developed a kinetic-stoichiometric model that indicates that the identified synthetic shunts could potentially enhance carbon fixation rate across the physiological range of irradiation and CO2, even if most of their enzymes operate at a tenth of Rubisco's maximal carboxylation activity. Glycolate reduction to glycolaldehyde is essential for several of the synthetic shunts but is not known to occur naturally. We, therefore, used computational design and directed evolution to establish this activity in two sequential reactions. An acetyl-CoA synthetase was engineered for higher stability and glycolyl-CoA synthesis. A propionyl-CoA reductase was engineered for higher selectivity for glycolyl-CoA and for use of NADPH over NAD+, thereby favoring reduction over oxidation. The engineered glycolate reduction module was then combined with downstream condensation and assimilation of glycolaldehyde to ribulose 1,5-bisphosphate, thus providing proof of principle for a carbon-conserving photorespiration pathway.

Quantum chemistry reveals thermodynamic principles of redox biochemistry.

Thermodynamics dictates the structure and function of metabolism. Redox reactions drive cellular energy and material flow. Hence, accurately quantifying the thermodynamics of redox reactions should reveal design principles that shape cellular metabolism. However, only few redox potentials have been measured, and mostly with inconsistent experimental setups. Here, we develop a quantum chemistry approach to calculate redox potentials of biochemical reactions and demonstrate our method predicts experimentally measured potentials with unparalleled accuracy. We then calculate the potentials of all redox pairs that can be generated from biochemically relevant compounds and highlight fundamental trends in redox biochemistry. We further address the question of why NAD/NADP are used as primary electron carriers, demonstrating how their physiological potential range fits the reactions of central metabolism and minimizes the concentration of reactive carbonyls. The use of quantum chemistry can revolutionize our understanding of biochemical phenomena by enabling fast and accurate calculation of thermodynamic values.

Artificial pathway emergence in central metabolism from three recursive phosphoketolase reactions.

The promiscuous activities of a recursive, generalist enzyme provide raw material for the emergence of metabolic pathways. Here, we use a synthetic biology approach to recreate such an evolutionary setup in central metabolism and explore how cellular physiology adjusts to enable recursive catalysis. We generate an Escherichia coli strain deleted in transketolase and glucose 6-phosphate dehydrogenase, effectively eliminating the native pentose phosphate pathway. We demonstrate that the overexpression of phosphoketolase restores prototrophic growth by catalyzing three consecutive reactions, cleaving xylulose 5-phosphate, fructose 6-phosphate, and, notably, sedoheptulose 7-phosphate. We find that the activity of the resulting synthetic pathway becomes possible due to the recalibration of steady-state concentrations of key metabolites, such that the in vivo cleavage rates of all three phosphoketolase substrates are similar. This study demonstrates our ability to rewrite one of nature's most conserved pathways and provides insight into the flexibility of cellular metabolism during pathway emergence.

Hydrocarbons Are Essential for Optimal Cell Size, Division, and Growth of Cyanobacteria.

Cyanobacteria are intricately organized, incorporating an array of internal thylakoid membranes, the site of photosynthesis, into cells no larger than other bacteria. They also synthesize C15-C19 alkanes and alkenes, which results in substantial production of hydrocarbons in the environment. All sequenced cyanobacteria encode hydrocarbon biosynthesis pathways, suggesting an important, undefined physiological role for these compounds. Here, we demonstrate that hydrocarbon-deficient mutants of Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 exhibit significant phenotypic differences from wild type, including enlarged cell size, reduced growth, and increased division defects. Photosynthetic rates were similar between strains, although a minor reduction in energy transfer between the soluble light harvesting phycobilisome complex and membrane-bound photosystems was observed. Hydrocarbons were shown to accumulate in thylakoid and cytoplasmic membranes. Modeling of membranes suggests these compounds aggregate in the center of the lipid bilayer, potentially promoting membrane flexibility and facilitating curvature. In vivo measurements confirmed that Synechococcus sp. PCC 7002 mutants lacking hydrocarbons exhibit reduced thylakoid membrane curvature compared to wild type. We propose that hydrocarbons may have a role in inducing the flexibility in membranes required for optimal cell division, size, and growth, and efficient association of soluble and membrane bound proteins. The recent identification of C15-C17 alkanes and alkenes in microalgal species suggests hydrocarbons may serve a similar function in a broad range of photosynthetic organisms.

Structure of the dual-function fructose-1,6/sedoheptulose-1,7-bisphosphatase from Thermosynechococcus elongatus bound with sedoheptulose-7-phosphate.

The dual-function fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) in cyanobacteria carries out two activities in the Calvin cycle. Structures of this enzyme from the cyanobacterium Synechocystis sp. PCC 6803 exist, but only with adenosine monophosphate (AMP) or fructose-1,6-bisphosphate and AMP bound. The mechanisms which control both selectivity between the two sugars and the structural mechanisms for redox control are still unresolved. Here, the structure of the dual-function FBP/SBPase from the thermophilic cyanobacterium Thermosynechococcus elongatus is presented with sedoheptulose-7-phosphate bound and in the absence of AMP. The structure is globally very similar to the Synechocystis sp. PCC 6803 enzyme, but highlights features of selectivity at the active site and loop ordering at the AMP-binding site. Understanding the selectivity and control of this enzyme is critical for understanding the Calvin cycle in cyanobacteria and for possible biotechnological application in plants.

Contribution of cyanobacterial alkane production to the ocean hydrocarbon cycle.

Hydrocarbons are ubiquitous in the ocean, where alkanes such as pentadecane and heptadecane can be found even in waters minimally polluted with crude oil. Populations of hydrocarbon-degrading bacteria, which are responsible for the turnover of these compounds, are also found throughout marine systems, including in unpolluted waters. These observations suggest the existence of an unknown and widespread source of hydrocarbons in the oceans. Here, we report that strains of the two most abundant marine cyanobacteria, Prochlorococcus and Synechococcus, produce and accumulate hydrocarbons, predominantly C15 and C17 alkanes, between 0.022 and 0.368% of dry cell weight. Based on global population sizes and turnover rates, we estimate that these species have the capacity to produce 2-540 pg alkanes per mL per day, which translates into a global ocean yield of ∼ 308-771 million tons of hydrocarbons annually. We also demonstrate that both obligate and facultative marine hydrocarbon-degrading bacteria can consume cyanobacterial alkanes, which likely prevents these hydrocarbons from accumulating in the environment. Our findings implicate cyanobacteria and hydrocarbon degraders as key players in a notable internal hydrocarbon cycle within the upper ocean, where alkanes are continually produced and subsequently consumed within days. Furthermore we show that cyanobacterial alkane production is likely sufficient to sustain populations of hydrocarbon-degrading bacteria, whose abundances can rapidly expand upon localized release of crude oil from natural seepage and human activities.

Pediatric tracheostomy: a 13-year experience.

Pediatric tracheostomy has been reported to be a surgical procedure with significant morbidity and mortality. The use of tracheostomy in airway management has changed over time as regards indication and outcome. A review of the last 13 years' experience in our institution was carried out to focus on this group of patients and the recent trends in airway management. A retrospective analysis of hospital records was done and information collected with respect to age, gender, indication for tracheostomy, duration, complications, and follow-up. Thirty-nine tracheotomies were done in 36 patients, of whom males outnumbered females 2:1. The mean patient age was 41.6 months while nearly a third were newborns. The indications were congenital and acquired obstructive lesions. Apart from nine cases, all have been treated and decannulated. Follow-up ranged from 1 month to 8 years, and decannulation time from 48 h to 45 months. Home tracheostomy care was very well managed by the parents. One tracheostomy-related death was encountered. Complications were minor and transient and occurred post-decannulation in our series, in contrast to the major complications, both acute and chronic, reported in the literature. More neonates and infants are undergoing tracheostomy and surviving. Pediatric tracheostomy is a safe procedure with home care by parents feasible.

Manometric evaluation of the intrathoracic stomach after gastric transposition in children.

Gastric transposition (GT) is one of the options for the esophageal replacement in children with esophageal atresia with or without tracheoesophageal fistula (EATEF). To date, no manometric studies have been conducted on the intrathoracic stomach after GT in EATEF patients; hence, this study was designed. Babies ( n=18) of EATEF who underwent esophageal replacement by GT were studied and manometry was correlated with the clinical outcome, age at surgery, and route of GT. The mean age at evaluation was 30.5 months (range 4-84 months). These cases were sub-stratified into group I (GT during neonatal period) and group II (GT during post-neonatal period). Mean age at surgery was 6 days and 7.8 months in groups I and II, respectively. There was no propulsive antegrade propagated peristaltic waves in any of the patients. Mean resting pressure and mean peak pressures were 19.5 and 50.4 mm Hg in groups I and II, respectively. Mass contractions to liquid swallow was noted in 77 and 55% of patients in groups I and II, respectively. There was no significant difference in the pressure parameters or appearance of mass contractions between group-I and group-II patients. Similarly, there was no significant difference in pressure parameters or appearance of mass contractions between the children who had transhiatal vs retrosternal GT. It needs to be determined whether the mass contractions noted in GT ever progress to a coordinated propulsive rhythmic contractions and whether this has a final bearing on the long-term functional outcome of GT patients.

Exstrophy variants: should they be considered malformation complexes separate from classic exstrophy?

PURPOSE: Exstrophy variants are very rare and have a better prognosis than classical exstrophy. The authors came across a case of superior vesical fissure (SVF) together with esophageal atresia and tracheoesophageal fistula (EATEF) and a case of SVF with gross limb anomalies. These associated malformations have not been reported so far in the literature and hence we reviewed all the cases of exstrophy variants presented to us with particular emphasis on the associated malformations. METHODS: Records (n=9) of patients who were diagnosed as exstrophy variants at our institution between 1989 and 2000 were evaluated retrospectively. RESULTS: Out of 9 cases, 7 cases had associated malformations: EATEF, urethral atresia, absent radius, large umbilical hernia, low anorectal malformation, true diphallus with bifid scrotum, or high anorectal malformation. CONCLUSION: The high incidence of associated congenital malformations, noted in our exstrophy variant series, raises doubts about the clubbing together of the exstrophy variants with classical exstrophy. Further investigation of such cases may elucidate shared or unique causes of the dysembryogenic mechanisms in the etiologies of variants of bladder exstrophy.

Urinary-tract infection affects somatic growth in unilateral symptomatic hydronephrosis.

To assess whether symptomatic unilateral ureteropelvic junction obstruction (SUUPJO) affects somatic growth and, if so, the parameters associated with it, 61 children (54 boys and 7 girls) who underwent pyeloplasty for SUUPJO without any other associated urological abnormalities were retrospectively studied. Height was compared with standard growth charts and was considered to be affected if it was below 2.00 Z-score. Such children were considered group B and the rest group A. Mean (+/-SD) age at presentation and mean (+/-SD) split renal function (SRF) (%) of the affected kidney were 6.0 +/- 4.0 years and 27.3 +/- 13.2, respectively, for the entire group. Somatic growth was affected in 16 (12 boys, 4 girls) children (26.2%). Urinary tract infection (UTI) was the presenting symptom in 11 (69%) and 5 (11%) children in groups B and A, respectively. Impaired somatic growth had no association with age at presentation or SRF, but a significant association (P < 0.001) was found with UTI. The mean post-surgery height percentile (2.92 +/- 4.85) over a mean follow-up of 3.37 +/- 1.86 years was significantly (P < 0.005) better compared with pre-surgery height percentile (0.67 +/- 0.96) in group B, indicating catch-up growth after surgery. In SUUPJO, somatic growth is affected. Presentation with UTI has a significant association, and height significantly improves after surgery in these patients.

Pediatric surgery in India - a specialty come of age?

The quality of neonatal surgical care and scientific publications are reliable yardsticks that were used to assess the status of pediatric surgery in India. A specific questionnaire to assess neonatal care and surgical outcome was mailed to all institutes imparting pediatric surgery training. Data were obtained regarding the outcome of important neonatal surgical conditions for the year 1998 and a PubMed literature search was performed to identify scientific articles between 1995 and 2000. Though a literature search was done to compile a complete list of publications of all the consultants in all the institutes, of the 24 questionnaires mailed, only 11 (45.8%) institutes provided data. The mean (range) annual neonatal admissions in neonatal surgical units was 137 (42-263). The mean newborn admissions requiring surgical intervention per surgeon per year was 36 (17-80). The overall survival was 57.2% (30%-75%), 70.8% (40%-100%), 90.4% (75%-100%), 74.7% (30%-100%), and 59.1% (0%-100%) for esophageal atresia (EA) with or without tracheoesophageal fistula (TEF), congenital diaphragmatic hernia, anorectal malformations, intestinal atresia, and abdominal-wall defects, respectively. The center that had the lowest survival in EA/TEF and CDH had the highest workload per consultant. Between 1995 and 2000, the mean number of scientific articles published in indexed journals compiled from all the institutes (n = 24) was 10.7 (0-84). In conclusion, this is a preliminary study toward setting up national databases of neonatal surgery in different parts of the world to set goals for improvement.

Giant congenital solitary cyst of the liver: report of a case.

Giant solitary nonparasitic cysts of the liver are rarely encountered in children, and establishing a preoperative diagnosis is usually difficult, especially when the cyst occupies the entire abdomen. We report herein the case of an 8-year-old girl found to have a giant congenital solitary cyst of the liver masquerading as an ovarian cyst.