1,084 research outputs found
In conversation with Nigel Scrutton
No full textNigel Scrutton FRS is Professor of Molecular Enzymology and Biophysical Chemistry at the University of Manchester and former Director of the Manchester Institute of Biotechnology (MIB). He obtained a first‐class degree in Biochemistry from King’s College London and followed this with a PhD at the University of Cambridge. His doctoral research, undertaken in Richard Perham’s laboratory, yielded fundamental breakthroughs in enzyme redesign that have stood the test of time. Nigel was awarded a ScD degree by the University of Cambridge in 2003. After faculty positions at the University of Leicester, Nigel was appointed Professor at the University of Manchester in 2005. Over the last 15 years, he has cemented his reputation as a world leader in the fields of enzyme engineering and biocatalysis, synthetic biology, biophysics and biomanufacturing, notably by establishing and directing the Synthetic Biology Research Centre ‘SYNBIOCHEM’ and UK Future Biomanufacturing Research Hub. In recognition of his scientific contributions, he has received many academic awards and accolades, including being elected as Fellow of the Royal Society earlier this year. In this interview, he highlights how fundamental studies of enzymatic catalysis and mechanisms are driving key advances in biotechnology and biomanufacturing, and describes how the experiences and mentors of his formative years helped to shape his successful career at the interface between discovery and application‐focused science
Multi-fragment DNA assembly of biochemical pathways via automated Ligase Cycling Reaction
No full textThe microbial production of commodity, fine and specialty chemicals and biofuels is a driving force in biotechnology. To do this, biochemical pathways to the target compound(s) must be deduced, suitable enzymes selected and then the genetic pathways must be designed and built for in vivo activity. The genetic design is crucial for balancing the pathway in vivo through regulation of transcription and translation but the possible permutations quickly generates a vast design space. Traditionally pathway assembly has been time-consuming and laborious but the advent of multi-fragment DNA assembly technologies has facilitated the possibility of multiplexed pathway construction allowing an increased capability to sample the design space. Furthermore, the implementation of laboratory automation allows error-reduced, high-throughput (HTP) construction of pathways. In this chapter we present an automated workflow that combines in silico design of DNA parts followed by pathway assembly using the Ligase Cycling Reaction (LCR) on robotics platforms, to allow multiplexed assembly of plasmid-borne gene pathways with high efficiency. The workflow begins with the design of DNA part sequences considering biological issues and ensuring compatibility with DNA synthesis and LCR assembly. Subsequently a laboratory protocol for HTP pathway assembly and screening is detailed allowing the production of over 96 plasmids simultaneously with a success rate of over 40 %. This workflow is easy to modify for other laboratories and will help to accelerate synthetic biology for diverse application
The causative role and therapeutic potential of the kynurenine pathway in neurodegenerative disease
No full textMetabolites of the kynurenine pathway (KP), which arise from the degradation of tryptophan, have been studied in detail for over a century and garnered the interest of the neuroscience community in the late 1970s and early 1980s with work uncovering the neuromodulatory potential of this pathway. Much research in the following decades has found that perturbations in the levels of KP metabolites likely contribute to the pathogenesis of several neurodegenerative diseases. More recently, it has become apparent that targeting KP enzymes, in particular kynurenine 3-monooxygenase (KMO), may hold substantial therapeutic potential for these disorders. Here we provide an overview of the KP, the neuroactive properties of KP metabolites and their role in neurodegeneration. We also discuss KMO as a therapeutic target for these disorders, and our recent resolution of the crystallographic structure of KMO, which will permit the development of new and improved KMO inhibitors which may ultimately expedite clinical application of these compounds. © 2013 Springer-Verlag Berlin Heidelberg
Also By The Same Author: AKTiveAuthor, a Citation Graph Approach to Name Disambiguation
No full textThe desire for definitive data and the semantic web drive for inference over heterogeneous data sources requires co-reference resolution to be performed on those data. In particular, name disambiguation is required to allow accurate publication lists, citation counts and impact measures to be determined. This paper describes a graph-based approach to author disambiguation on large-scale citation networks. Using self-citation, co-authorship and document source analyses, AKTiveAuthor clusters papers, achieving precision of 0.997 and recall of 0.818 over a test group of eight surname clusters
Incorporation of hydrostatic pressure into models of hydrogen tunneling highlights a role for pressure-modulated promoting vibrations
No full textHydrostatic pressure offers an alternative to temperature as an experimental probe of hydrogen-transfer reactions. H tunneling reactions have been shown to exhibit kinetic isotope effects (KIEs) that are sensitive to pressure, and environmentally coupled H tunneling reactions, those reactions in which H transfer is coupled to atomic fluctuations (a promoting vibration) along the reaction coordinate, often have quite temperature-dependent KIEs. We present here a theoretical treatment of the combined effect of temperature and pressure on environmentally coupled H tunneling reactions. We develop a generalized expression for the KIE, which can be used as a simple fitting function for combined experimental temperature- and pressure-dependent KIE data sets. With this expression, we are able to extract information about the pressure dependence of both the apparent tunneling distance and the frequency of the promoting vibration. The KIE expression is tested on two data sets {the reduction of chloranil by leuco crystal violet [Isaacs, N. S., Javaid, K., and Rannala, E. (1998) J. Chem. Soc., Perkin Trans. 2, 709-711] and the reduction of morphinone reductase by NADH [Hay, S., Sutcliffe, M. J., and Scrutton, N. S. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 507-512]} and suggests that hydrostatic pressure is a sensitive probe of nuclear quantum mechanical effects in H-transfer reactions. © 2008 American Chemical Society
Incorporation of Hydrostatic Pressure into Models of Hydrogen Tunneling Highlights a Role for Pressure-Modulated Promoting Vibrations
No full textHydrostatic pressure offers an alternative to temperature as an experimental probe of hydrogen-transfer reactions. H tunneling reactions have been shown to exhibit kinetic isotope effects (KIEs) that are sensitive to pressure, and environmentally coupled H tunneling reactions, those reactions in which H transfer is coupled to atomic fluctuations (a promoting vibration) along the reaction coordinate, often have quite temperature-dependent KIEs. We present here a theoretical treatment of the combined effect of temperature and pressure on environmentally coupled H tunneling reactions. We develop a generalized expression for the KIE, which can be used as a simple fitting function for combined experimental temperature- and pressure-dependent KIE data sets. With this expression, we are able to extract information about the pressure dependence of both the apparent tunneling distance and the frequency of the promoting vibration. The KIE expression is tested on two data sets {the reduction of chloranil by leuco crystal violet [Isaacs, N. S., Javaid, K., and Rannala, E. (1998) J. Chem. Soc., Perkin Trans. 2, 709−711] and the reduction of morphinone reductase by NADH [Hay, S., Sutcliffe, M. J., and Scrutton, N. S. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 507−512]} and suggests that hydrostatic pressure is a sensitive probe of nuclear quantum mechanical effects in H-transfer reactions
The dimeric form of flavocytochrome P450 BM3 is catalytically functional as a fatty acid hydroxylase
No full textIn the model P450 BM3 system, the P450 is fused to its diflavin reductase partner in a single polypeptide. BM3 dimerizes in solution, but the catalytic relevance of the phenomenon was hitherto unknown. We show that BM3 fatty acid hydroxylase specific activity decreases sharply at low enzyme concentrations, consistent with separation of active dimer into inactive monomer. Reductase-dependent specific activities are maintained or enhanced at low concentration, suggesting inter-flavin electron transfer is unaffected. Fatty acid oxidation is reconstituted by mixing inactive oxygenase (A264H) and FMN-depleted (G570D) mutants, demonstrating that inter-monomer (FMN1-to-heme2) electron transfer supports oxygenase activity in the BM3 dimer
Flavocytochrome P450 BM3 substrate selectivity and electron transfer in a model cytochrome P450
No full textBeyond the Historical Perspective on Hydrogen and Electron Transfers
No full textA brief overview of proton and electron transfer history is given, and various
features influencing enzymatic catalysis are discussed. Examples of generic
behavior are considered, together with questions that can be addressed for both
experimental and computational results. Examples of high and low pre-exponential
factors A of the intrinsic rate constant k_H ranging from ~10^(17) s^(-1) to ~10^4 s^(-1) and normal (~10^(13)) are noted with significant error bars and
discussed
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