Crystal structure and substrate specificity of the thermophilic serine:pyruvate aminotransferase from Sulfolobus solfataricus.

Journal ArticleResearch Support, Non-U.S. Gov'tThe three-dimensional structure of the Sulfolobus solfataricus serine:pyruvate aminotransferase has been determined to 1.8 Å resolution. The structure of the protein is a homodimer that adopts the type I fold of pyridoxal 5'-phosphate (PLP)-dependent aminotransferases. The structure revealed the PLP cofactor covalently bound in the active site to the active-site lysine in the internal aldimine form. The structure of the S. solfataricus enzyme was also determined with an amino form of the cofactor pyridoxamine 5'-phosphate bound in the active site and in complex with gabaculine, an aminotransferase inhibitor. These structures showed the changes in the enzyme active site during the course of the catalytic reaction. A comparison of the structure of the S. solfataricus enzyme with that of the closely related alanine:glyoxylate aminotransferase has identified structural features that are proposed to be responsible for the differences in substrate specificity between the two enzymes. These results have been complemented by biochemical studies of the substrate specificity and thermostability of the S. solfataricus enzyme.University of ExeterBBSRCEPSRCWellcome Trus

Engineering a Seven Enzyme Biotransformation using Mathematical Modelling and Characterized Enzyme Parts (article)

This is the final version. Available on open access from Wiley via the DOI in this recordThe dataset associated with this article is located in ORE at: https://doi.org/10.24378/exe.1623Multi‐step enzyme reactions offer considerable cost and productivity benefits. Process models offer a route to understanding the complexity of these reactions, and allow for their optimization. Despite the increasing prevalence of multi‐step biotransformations, there are few examples of process models for enzyme reactions. From a toolbox of characterized enzyme parts, we demonstrate the construction of a process model for a seven enzyme, three step biotransformation using isolated enzymes. Enzymes for cofactor regeneration were employed to make this in vitro reaction economical. Good modelling practice was critical in evaluating the impact of approximations and experimental error. We show that the use and validation of process models was instrumental in realizing and removing process bottlenecks, identifying divergent behavior, and for the optimization of the entire reaction using a genetic algorithm. We validated the optimized reaction to demonstrate that complex multi‐step reactions with cofactor recycling involving at least seven enzymes can be reliably modelled and optimized.Biotechnology & Biological Sciences Research Council (BBSRC)GlaxoSmithKlin

Complexity reduction and opportunities in the design, integration and intensification of biocatalytic processes for metabolite synthesis

This is the final version. Available on open access from Frontiers Media via the DOI in this recordThe biosynthesis of metabolites from available starting materials is becoming an ever important area due to the increasing demands within the life science research area. Access to metabolites is making essential contributions to analytical, diagnostic, therapeutic and different industrial applications. These molecules can be synthesized by the enzymes of biological systems under sustainable process conditions. The facile synthetic access to the metabolite and metabolite-like molecular space is of fundamental importance. The increasing knowledge within molecular biology, enzyme discovery and production together with their biochemical and structural properties offers excellent opportunities for using modular cell-free biocatalytic systems. This reduces the complexity of synthesizing metabolites using biological whole-cell approaches or by classical chemical synthesis. A systems biocatalysis approach can provide a wealth of optimized enzymes for the biosynthesis of already identified and new metabolite molecules

Micro-enterprises: small enough to care?

This report presents findings of an evaluation of micro-enterprises in social care in England, which ran from 2013 to 2015. Organisations are here classed as micro if they employ five or fewer full-time equivalent staff. The aim of the project was to test the extent to which micro-enterprises deliver services that are personalised, valued, innovative and cost-effective, and how they compare with small, medium and large providers. Working in three parts of the country, researchers compared 27 organisations providing care and support, of which 17 were micro-enterprises, 2 were small, 4 were medium and 4 were large. The project team interviewed and surveyed 143 people (staff, older people, people with disabilities and carers) who received support from the 27 providers. The findings presented are relevant to people who use services and their families; social care commissioners; regulators and policy makers at a local and national level; people who provide care services; and social entrepreneurs who are considering setting up micro forms of support. The research was based at the University of Birmingham. It was funded by the Economic and Social Research Council (ESRC), as part of a project entitled Does Smaller mean Better? Evaluating Micro-enterprises in Adult Social Care (ESRC Standard Grant ES/K002317/1)

An NADPH-Dependent Genetic Switch Regulates Plant Infection by the Rice Blast Fungus

To cause rice blast disease, the fungus Magnaporthe oryzae breaches the tough outer cuticle of the rice leaf by using specialized infection structures called appressoria. These cells allow the fungus to invade the host plant and proliferate rapidly within leaf tissue. Here, we show that a unique NADPH-dependent genetic switch regulates plant infection in response to the changing nutritional and redox conditions encountered by the pathogen. The biosynthetic enzyme trehalose-6-phosphate synthase (Tps1) integrates control of glucose-6-phosphate metabolism and nitrogen source utilization by regulating the oxidative pentose phosphate pathway, the generation of NADPH, and the activity of nitrate reductase. We report that Tps1 directly binds to NADPH and, thereby, regulates a set of related transcriptional corepressors, comprising three proteins, Nmr1, Nmr2, and Nmr3, which can each bind NADP. Targeted deletion of any of the Nmr-encoding genes partially suppresses the nonpathogenic phenotype of a Δtps1 mutant. Tps1-dependentNmr corepressors control the expression of a set of virulence-associated genes that are derepressed during appressorium-mediated plant infection. When considered together, these results suggest that initiation of rice blast disease by M. oryzae requires a regulatory mechanism involving an NADPH sensor protein, Tps1, a set of NADP-dependent transcriptional corepressors, and the nonconsuming interconversion ofNADPHandNADPacting as signal transducer

Keratan sulfate phenotype in the β-1,3-N-acetylglucosaminyltransferase-7-null mouse cornea

Purpose: Synthesis of keratan sulfate (KS) relies on coordinated action of multiple enzymes, including the N-acetylglucosamine–transferring enzyme, β-1,3-N-acetylglucosaminyltransferase-7 (β3GnT7). A mouse model deficient in β3GnT7 was developed to explore structural changes in KS and the extracellular matrix (ECM; i.e., the corneal stroma), elucidate the KS biosynthesis mechanism, and understand its role in corneal organization. Methods: A knockout vector for the β3GnT7-encoding gene, B3gnt7, was created to develop heterozygous- (htz) and homozygous-null (null) knockouts. Epithelial, stromal, and whole cornea thicknesses were measured from each group. Proteoglycans were stained with cupromeronic blue for visualization by electron microscopy, and Western blot analyses were conducted on the KS core protein, lumican. Corneal sections were labelled fluorescently for KS and chondroitin sulfate/dermatan sulfate (CS/DS) using monoclonal antibodies 1B4 or 2B6, respectively. Results: Wild-type (WT) and htz corneas were of similar stromal thickness, whereas null specimens measured relatively thin. Electron micrographs revealed that WT and htz samples contained comparable levels of KS- and CS/DS-PGs. Null corneas, however, lacked detectable KS and featured uncharacteristically elongated electron dense PG filaments, which were susceptible to chondroitinase ABC digestion. Western blotting revealed lumican in the null corneas was substituted with low-molecular-weight KS, relative to WT or htz tissue. KS was not immunohistochemically detectable in the null cornea, whereas CS/DS content appeared increased. Conclusions: Addition of N-acetylglucosamine via β3GnT7 to KS glycosaminoglycans is necessary for their biosynthesis. Without β3GnT7, murine corneal stromas lack KS and appear to compensate for this loss with upregulation of chondroitinase ABC-sensitive PGs

Structural and biochemical characterisation of Archaeoglobus fulgidus esterase reveals a bound CoA molecule in the vicinity of the active site

A new carboxyl esterase, AF-Est2, from the hyperthermophilic archaeon Archaeoglobus fulgidus has been cloned, over-expressed in Escherichia coli and biochemically and structurally characterized. The enzyme has high activity towards short- to medium-chain pnitrophenyl carboxylic esters with optimal activity towards the valerate ester. The AF-Est2 has good solvent and pH stability and is very thermostable, showing no loss of activity after incubation for 30 min at 80 °C. The 1.4 Å resolution crystal structure of AF-Est2 reveals Coenzyme A (CoA) bound in the vicinity of the active site. Despite the presence of CoA bound to the AF-Est2 this enzyme has no CoA thioesterase activity. The pantetheine group of CoA partially obstructs the active site alcohol pocket suggesting that this ligand has a role in regulation of the enzyme activity. A comparison with closely related α/β hydrolase fold 2 enzyme structures shows that the AF-Est2 has unique structural features that allow CoA binding. A comparison of the structure of AF-Est2 with the human carboxyl esterase 1, which has CoA thioesterase activity, reveals that CoA is bound to different parts of the core domain in these two enzymes and approaches the active site from opposite directions.This work was supported by the Hotzyme project (grant agreement no. 265933) financed by the European Union 7th Framework Programme FP7/2007-2013. WF is funded by a BBSRC PhD studentship. MI would like to thank the BBSRC funded ERA-IB grant BB/L002035/1 and the University of Exeter for support. The authors would like to thank the Diamond Synchrotron Light Source for access to beamline I03 (proposals No. MX8889 and No. MX11945) and the beamline scientists for assistance. The work of ML was funded by the Graduate School VLAG Wageningen, the Netherlan

Methodological issues in testing the marginal productivity theory

Previous tests of the marginal productivity theory have been criticized on several grounds reviewed by the authors. One important deficiency has been the small number of factor inputs entered in the production functions. In 1978 Gottschalk suggested a method to estimate production functions with many inputs by assuming that the production process can be split into subprocesses. This reduces the probability of multicollinearity. The authors show that the method depends on an additional assumption. Tinbergen has developed a method for avoiding this assumption. Its application to American cross-section (state) data did not alter the estimated coefficients greatly

Reflections and Experiences of a Co-Researcher involved in a Renal Research Study

Background Patient and Public Involvement (PPI) is seen as a prerequisite for health research. However, current Patient and public involvement literature has noted a paucity of recording of patient and public involvement within research studies. There have been calls for more recordings and reflections, specifically on impact. Renal medicine has also had similar criticisms and any reflections on patient and public involvement has usually been from the viewpoint of the researcher. Roles of patient and public involvement can vary greatly from sitting on an Advisory Group to analysing data. Different PPI roles have been described within studies; one being a co-researcher. However, the role of the co-researcher is largely undefined and appears to vary from study to study. Methods The aims of this paper are to share one first time co-researcher's reflections on the impact of PPI within a mixed methods (non-clinical trial) renal research study. A retrospective, reflective approach was taken using data available to the co-researcher as part of the day-to-day research activity. Electronic correspondence and documents such as meeting notes, minutes, interview thematic analysis and comments on documents were re-examined. The co-researcher led on writing this paper. Results This paper offers a broad definition of the role of the co-researcher. The co-researcher reflects on undertaking and leading on the thematic analysis of interview transcripts, something she had not previously done before. The co-researcher identified a number of key themes; the differences in time and responsibility between being a coresearcher and an Advisory Group member; how the role evolved and involvement activities could match the co-researchers strengths (and the need for flexibility); the need for training and support and lastly, the time commitment. It was also noted that it is preferable that a co-researcher needs to be involved from the very beginning of the grant application. Conclusions The reflections, voices and views of those undertaking PPI has been largely underrepresented in the literature. The role of co-researcher was seen to be rewarding but demanding, requiring a large time commitment. It is hoped that the learning from sharing this experience will encourage others to undertake this role, and encourage researchers to reflect on the needs of those involved.Peer reviewedFinal Published versio