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.

Engineering Nitrogenases for Synthetic Nitrogen Fixation: From Pathway Engineering to Directed Evolution.

Globally, agriculture depends on industrial nitrogen fertilizer to improve crop growth. Fertilizer production consumes fossil fuels and contributes to environmental nitrogen pollution. A potential solution would be to harness nitrogenases-enzymes capable of converting atmospheric nitrogen N2 to NH3 in ambient conditions. It is therefore a major goal of synthetic biology to engineer functional nitrogenases into crop plants, or bacteria that form symbiotic relationships with crops, to support growth and reduce dependence on industrially produced fertilizer. This review paper highlights recent work toward understanding the functional requirements for nitrogenase expression and manipulating nitrogenase gene expression in heterologous hosts to improve activity and oxygen tolerance and potentially to engineer synthetic symbiotic relationships with plants.

The Ycf48 accessory factor occupies the site of the oxygen-evolving manganese cluster during photosystem II biogenesis.

Robust oxygenic photosynthesis requires a suite of accessory factors to ensure efficient assembly and repair of the oxygen-evolving photosystem two (PSII) complex. The highly conserved Ycf48 assembly factor binds to the newly synthesized D1 reaction center polypeptide and promotes the initial steps of PSII assembly, but its binding site is unclear. Here we use cryo-electron microscopy to determine the structure of a cyanobacterial PSII D1/D2 reaction center assembly complex with Ycf48 attached. Ycf48, a 7-bladed beta propeller, binds to the amino-acid residues of D1 that ultimately ligate the water-oxidising Mn4CaO5 cluster, thereby preventing the premature binding of Mn2+ and Ca2+ ions and protecting the site from damage. Interactions with D2 help explain how Ycf48 promotes assembly of the D1/D2 complex. Overall, our work provides valuable insights into the early stages of PSII assembly and the structural changes that create the binding site for the Mn4CaO5 cluster.

Near ambient N2 fixation on solid electrodes versus enzymes and homogeneous catalysts.

The Mo/Fe nitrogenase enzyme is unique in its ability to efficiently reduce dinitrogen to ammonia at atmospheric pressures and room temperature. Should an artificial electrolytic device achieve the same feat, it would revolutionize fertilizer production and even provide an energy-dense, truly carbon-free fuel. This Review provides a coherent comparison of recent progress made in dinitrogen fixation on solid electrodes, homogeneous catalysts and nitrogenases. Specific emphasis is placed on systems for which there is unequivocal evidence that dinitrogen reduction has taken place. By establishing the cross-cutting themes and synergies between these systems, we identify viable avenues for future research.

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.

Versatile selective evolutionary pressure using synthetic defect in universal metabolism.

The non-natural needs of industrial applications often require new or improved enzymes. The structures and properties of enzymes are difficult to predict or design de novo. Instead, semi-rational approaches mimicking evolution entail diversification of parent enzymes followed by evaluation of isolated variants. Artificial selection pressures coupling desired enzyme properties to cell growth could overcome this key bottleneck, but are usually narrow in scope. Here we show diverse enzymes using the ubiquitous cofactors nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) can substitute for defective NAD regeneration, representing a very broadly-applicable artificial selection. Inactivation of Escherichia coli genes required for anaerobic NAD regeneration causes a conditional growth defect. Cells are rescued by foreign enzymes connected to the metabolic network only via NAD or NADP, but only when their substrates are supplied. Using this principle, alcohol dehydrogenase, imine reductase and nitroreductase variants with desired selectivity modifications, and a high-performing isopropanol metabolic pathway, are isolated from libraries of millions of variants in single-round experiments with typical limited information to guide design.

Static and Dynamic Postannealing Strategies for Roll-to-Roll Fabrication of DC Magnetron Sputtered Bismuth Telluride Thin Films onto Polymer Webs.

High-throughput roll-to-roll processes are desirable to scale up the manufacture of flexible thermoelectric generators. While vacuum deposition onto a heated dynamic substrate presents a considerable engineering challenge, viable postdeposition in-line annealing processes are considered as an alternative to improve the functional performance of as-deposited films. The effect of infrared and electron-beam irradiations of 1 µm thick bismuth telluride thin films, produced by a vacuum roll-to-roll process for use as thermoelectric materials, was examined. A static vacuum oven and pulsed high-energy electron beam were also studied as control groups. All annealing strategies increased the crystallite size and decreased the Te content. Only the static vacuum oven treatment was shown to significantly improve the film's crystallinity. After 1 h annealing, the power factor improved by 400% (from 2.8 to 14 × 10-4 W/mK2), which, to the knowledge of the authors, is the highest reported thermoelectric performance of postannealed or hot-deposited Bi-Te films. As for in-line annealing, infrared and electron-beam post treatments improved the power factor by 146% (from 2.8 to 6.9 × 10-4 W/mK2) and 64% (from 2.8 to 4.6 × 10-4 W/mK2), respectively.

Publisher Correction: Chlorophyll f synthesis by a super-rogue photosystem II complex.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Chlorophyll f synthesis by a super-rogue photosystem II complex.

Certain cyanobacteria synthesize chlorophyll molecules (Chl d and Chl f) that absorb in the far-red region of the solar spectrum, thereby extending the spectral range of photosynthetically active radiation1,2. The synthesis and introduction of these far-red chlorophylls into the photosynthetic apparatus of plants might improve the efficiency of oxygenic photosynthesis, especially in far-red enriched environments, such as in the lower regions of the canopy3. Production of Chl f requires the ChlF subunit, also known as PsbA4 (ref. 4) or super-rogue D1 (ref. 5), a paralogue of the D1 subunit of photosystem II (PSII) which, together with D2, bind cofactors involved in the light-driven oxidation of water. Current ideas suggest that ChlF oxidizes Chl a to Chl f in a homodimeric ChlF reaction centre (RC) complex and represents a missing link in the evolution of the heterodimeric D1/D2 RC of PSII (refs. 4,6). However, unambiguous biochemical support for this proposal is lacking. Here, we show that ChlF can substitute for D1 to form modified PSII complexes capable of producing Chl f. Remarkably, mutation of just two residues in D1 converts oxygen-evolving PSII into a Chl f synthase. Overall, we have identified a new class of PSII complex, which we term 'super-rogue' PSII, with an unexpected role in pigment biosynthesis rather than water oxidation.

Developing highly nanoporous titanate structures via wet chemical conversion of DC magnetron sputtered titanium thin films.

Titanate structures have been widely investigated as biomedical component surfaces due to their bioactive, osteoinductive and antibacterial properties. However, these surfaces are limited to Ti and its alloys, due to the nature of the chemical conversion employed. The authors present a new method for generating nanoporous titanate structures on alternative biomaterial surfaces, such as other metals/alloys, ceramics and polymers, to produce bioactive and/or antibacterial properties in a simple yet effective way. Wet chemical (NaOH; 5 M; 60 °C; 24 h) conversion of DC magnetron sputtered Ti surfaces on 316L stainless steel were investigated to explore effects of microstructure on sodium titanate conversion. It was found that the more equiaxed thin films (B/300) generated the thickest titanate structures (ca. 1.6 µm), which disagreed with the proposed hypothesis of columnar structures allowing greater NaOH ingress. All film parameters tested ultimately generated titanate structures, as confirmed via EDX, SEM, XPS, XRD, FTIR and Raman analyses. Additionally, the more columnar structures (NB/NH & B/NH) had a greater quantity of Na (ca. 26 at.%) in the top portion of the films, as confirmed via XPS, however, on average the Na content was consistent across the films (ca. 5-9 at.%). Film adhesion for the more columnar structures (ca. 42 MPa), even on polished substrates, were close to that of the FDA requirement for plasma-sprayed HA coatings (ca. 50 MPa). This study demonstrates the potential of these surfaces to be applied onto a wide variety of material types, even polymeric materials, due to the lower processing temperatures utilised, with the vision to generate bioactive and/or antibacterial properties on a plethora of bioinert materials.

Thermal Activation of Electrochemical Seed Surfaces for Selective and Tunable Hydrophobic Patterning.

Remarkable interfacial behaviors are observed in nature. Our efforts, directed toward replicating the structures, chemistries, and therefore functional properties of natural nonwetting surfaces, are competing with the result of billions of years of natural selection. The application of man-made surfaces is challenged by their poor longevity in aggressive environmental or applied service conditions. This study reports on a new approach for the creation of multiscale hierarchical surface patterns in metals, which exploits thermodynamic phenomena in advanced manufacturing processes. While hydrophobic coatings can be produced with relative ease by electrodeposition, these fractal-type structures tend to have poor structural integrity and hence are not durable. In this method, "seed surfaces" are directly written onto substrates by selective electrodeposition, after which they are irradiated by a large-area, pulsed electron beam to invoke a beading phenomenon, which is studied here. The length scale of these beads is shown to depend upon the melt time of the liquid metal. The created surfaces are shown to yield high water contact angles (145°) without subsequent chemical modification, and high adhesion properties reminiscent of the "rose petal" hydrophobic effect. The size and morphology and hence the hydrophobic effect of the surface beads generated are correlated with the thickness of the electrodeposited coating and hence the melt lifetime upon electron irradiation. This new rapid approach for tunable hydrophobic surface creation has applications for developing precision hydrophobic patterns and is insensitive to surface complexity.

A Sensitive Coupled Enzyme Assay for Measuring Kinase and ATPase Kinetics Using ADP-Specific Hexokinase.

Kinases and ATPases perform essential biological functions in metabolism and regulation. Activity of these enzymes is commonly measured by coupling ATP consumption to the synthesis of a detectable product. For most assay systems the ATP concentration during the reaction is unknown, compromising the precision of the assay. Using the ADP-specific hexokinase (ADP-HK) from the thermophilic archaeon Thermococcus litoralis the protocol outlined here allows real time coupling of ATP consumption to downstream signal change enabling accurate kinetic measurements. ADP-HK phosphorylates glucose that is then used by glucose-6-phosphate dehydrogenase to reduce NAD+ to NADH which can be measured at 340 nm. We have shown this assay to be sensitive to the detection of micromole quantities of ADP with no detectable background from ATP.

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.

Coping with strong translational noncrystallographic symmetry and extreme anisotropy in molecular replacement with Phaser: human Rab27a.

Data pathologies caused by effects such as diffraction anisotropy and translational noncrystallographic symmetry (tNCS) can dramatically complicate the solution of the crystal structures of macromolecules. Such problems were encountered in determining the structure of a mutant form of Rab27a, a member of the Rab GTPases. Mutant Rab27a constructs that crystallize in the free form were designed for use in the discovery of drugs to reduce primary tumour invasiveness and metastasis. One construct, hRab27aMut, crystallized within 24 h and diffracted to 2.82 Šresolution, with a unit cell possessing room for a large number of protein copies. Initial efforts to solve the structure using molecular replacement by Phaser were not successful. Analysis of the data set revealed that the crystals suffered from both extreme anisotropy and strong tNCS. As a result, large numbers of reflections had estimated standard deviations that were much larger than their measured intensities and their expected intensities, revealing problems with the use of such data at the time in Phaser. By eliminating extremely weak reflections with the largest combined effects of anisotropy and tNCS, these problems could be avoided, allowing a molecular-replacement solution to be found. The lessons that were learned in solving this structure have guided improvements in the numerical analysis used in Phaser, particularly in identifying diffraction measurements that convey very little information content. The calculation of information content could also be applied as an alternative to ellipsoidal truncation. The post-mortem analysis also revealed an oversight in accounting for measurement errors in the fast rotation function. While the crystal of mutant Rab27a is not amenable to drug screening, the structure can guide new modifications to obtain more suitable crystal forms.

Two step porosification of biomimetic thin-film hydroxyapatite/alpha-tri calcium phosphate coatings by pulsed electron beam irradiation.

Here we show a new and effective methodology for rapid/controllable porosification of thin-film ceramics, which may be applied in medical devices/electronics and membrane nano-filtration. Dense hydroxyapatite applied to Ti6Al4V by plasma-assisted PVD was electron-beam irradiated to induce flash melting/boiling. Deposited coatings contained amorphous and nano-crystalline/stoichiometric hydroxyapatite (~35 nm). Irradiation (voltages 13-29 kV) led to ablation (up to 45% mass loss) and average/maximum pore areas from (0.07-1.66)/(0.69-92.53) µm2, mimicking the human cortical bone. Vitrification above 1150 °C formed (~62-30 nm) crystallites of α-Tri Calcium Phosphate. Unique porosification resulted from irradiation-induced sub-surface boiling and limited thermal conductivity of hydroxyapatite, causing material to expand/explode through the more quickly solidified top surface. Commercially applicable, roughened Ti6Al4V exacerbated the heating and boiling explosion phenomenon in certain regions, producing an array of pore sizes. Scaffold-like morphologies were generated by interconnection of micron/sub-micron porosity, showing great potential for facile generation of a biomimetic surface treatment for osseointegration.

Ycf48 involved in the biogenesis of the oxygen-evolving photosystem II complex is a seven-bladed beta-propeller protein.

Robust photosynthesis in chloroplasts and cyanobacteria requires the participation of accessory proteins to facilitate the assembly and maintenance of the photosynthetic apparatus located within the thylakoid membranes. The highly conserved Ycf48 protein acts early in the biogenesis of the oxygen-evolving photosystem II (PSII) complex by binding to newly synthesized precursor D1 subunit and by promoting efficient association with the D2 protein to form a PSII reaction center (PSII RC) assembly intermediate. Ycf48 is also required for efficient replacement of damaged D1 during the repair of PSII. However, the structural features underpinning Ycf48 function remain unclear. Here we show that Ycf48 proteins encoded by the thermophilic cyanobacterium Thermosynechococcus elongatus and the red alga Cyanidioschyzon merolae form seven-bladed beta-propellers with the 19-aa insertion characteristic of eukaryotic Ycf48 located at the junction of blades 3 and 4. Knowledge of these structures has allowed us to identify a conserved "Arg patch" on the surface of Ycf48 that is important for binding of Ycf48 to PSII RCs but also to larger complexes, including trimeric photosystem I (PSI). Reduced accumulation of chlorophyll in the absence of Ycf48 and the association of Ycf48 with PSI provide evidence of a more wide-ranging role for Ycf48 in the biogenesis of the photosynthetic apparatus than previously thought. Copurification of Ycf48 with the cyanobacterial YidC protein insertase supports the involvement of Ycf48 during the cotranslational insertion of chlorophyll-binding apopolypeptides into the membrane.

Human Skeletal Stem Cell Response to Multiscale Topography Induced by Large Area Electron Beam Irradiation Surface Treatment.

The healthcare socio-economic environment is irreversibly changing as a consequence of an increasing aging population, consequent functional impairment, and patient quality of life expectations. The increasing complexity of ensuing clinical scenarios compels a critical search for novel musculoskeletal regenerative and replacement strategies. While joint arthroplasty is a highly effective treatment for arthritis and osteoporosis, further innovation and refinement of uncemented implants are essential in order to improve implant integration and reduce implant revision rate. This is critical given financial restraints and the drive to improve cost-effectiveness and quality of life outcomes. Multi-scale modulation of implant surfaces, offers an innovative approach to enhancement in implant performance. In the current study, we have examined the potential of large area electron beam melting to alter the surface nanotopography in titanium alloy (Ti6Al4V). We evaluated the in vitro osteogenic response of human skeletal stem cells to the resultant nanotopography, providing evidence of the relationship between the biological response, particularly Collagen type I and Osteocalcin gene activation, and surface nanoroughness. The current studies demonstrate osteogenic gene induction and morphological cell changes to be significantly enhanced on a topography Ra of ~40 nm with clinical implications therein for implant surface treatment and generation.