Gideon Grogan

The University of York, York, England, United Kingdom

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Publications (111)361.49 Total impact

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    ABSTRACT: Transaminases are valuable enzymes for industrial biocatalysis and enable the preparation of optically pure amines. For these transformations they require either an amine donor (amination of ketones) or an amine acceptor (deamination of racemic amines). Herein transaminases are shown to react with aromatic β-fluoroamines, thus leading to simultaneous enantioselective dehalogenation and deamination to form the corresponding acetophenone derivatives in the absence of an amine acceptor. A series of racemic β-fluoroamines was resolved in a kinetic resolution by tandem hydrodefluorination/deamination, thus giving the corresponding amines with up to greater than 99 % ee. This protocol is the first example of exploiting the catalytic promiscuity of transaminases as a tool for novel transformations.
    No preview · Article · Feb 2016 · Angewandte Chemie International Edition
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    ABSTRACT: Transaminases are valuable enzymes for industrial biocatalysis and enable the preparation of optically pure amines. For these transformations they require either an amine donor (amination of ketones) or an amine acceptor (deamination of racemic amines). Herein transaminases are shown to react with aromatic β-fluoroamines, thus leading to simultaneous enantioselective dehalogenation and deamination to form the corresponding acetophenone derivatives in the absence of an amine acceptor. A series of racemic β-fluoroamines was resolved in a kinetic resolution by tandem hydrodefluorination/deamination, thus giving the corresponding amines with up to greater than 99 % ee. This protocol is the first example of exploiting the catalytic promiscuity of transaminases as a tool for novel transformations.
    No preview · Article · Feb 2016 · Angewandte Chemie
  • Gideon Grogan · Nicholas J Turner
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    ABSTRACT: Imine reductases (IREDs) are NADPH-dependent oxidoreductases that catalyse the asymmetric reduction of cyclic prochiral imines to amines, with excellent stereoselectivity. Since their discovery, stereocomplementary IREDs have been applied to the production of both (S) and (R) cyclic secondary amines, and the expansion in gene sequences recently identified has hinted at new substrate ranges that extend into acyclic imines and even suggest the possibility of asymmetric reductive amination from suitable ketone and amine precursors. Structural studies of various IREDs are beginning to reveal the complexities inherent in determining substrate range, stereoselectivity and mechanism in these enzymes, which represent a valuable emerging addition to the toolbox of available biocatalysts for chiral amine production.
    No preview · Article · Dec 2015 · Chemistry - A European Journal
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    Full-text · Dataset · Dec 2015
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    ABSTRACT: The actinomycete Rhodococcus jostii RHA1 contains a multitude of oxygenase enzymes, consonant with its remarkable activities in the catabolism of hydrophobic xenobiotic compounds. In the interests of identifying activities for the transformation of drug molecules, we have cloned genes encoding 23 cytochrome P450 heme domains from R. jostii and expressed them as fusions with the P450 reductase domain (RhfRED) of cytochrome P450Rhf from Rhodococcus sp. NCIMB 9784. Fifteen of the fusions were expressed in the soluble fraction of Escherichia coli Rosetta (DE3) cells. Strains expressing the fusions of RhfRED with genes ro02604, ro04667, ro11069, ro11320, ro11277, ro08984 and ro04671 were challenged with 48 commercially available drugs revealing many different activities commensurate with P450-catalyzed hydroxylation and demethylation reactions. One recombinant strain, expressing the fusion of P450 gene ro11069 (CYP257A1) with RhfRED, and named Ro07-RhfRED, catalyzed the N-demethylation of diltiazem and imipramine. This observation was in accord with previous reports of this enzyme's activity as a demethylase of alkaloid substrates. Ro07-RhfRED was purified and characterised, and applied in cell-free biotransformations of imipramine (7μM) giving a 63% conversion to the N-desmethyl product. Copyright © 2015 Elsevier Ltd. All rights reserved.
    No preview · Article · Jul 2015 · Bioorganic & medicinal chemistry
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    ABSTRACT: Oxidoreductases from Streptomyces sp. GF3546 [3546-IRED], Bacillus cereus BAG3X2 (BcIRED) and Nocardiopsis halophila (NhIRED) each reduce prochiral 2-methylpyrroline (2MPN) to (S)-2-methylpyrrolidine with >95 % ee and also a number of other imine substrates with good selectivity. Structures of BcIRED and NhIRED have helped to identify conserved active site residues within this subgroup of imine reductases that have S selectivity towards 2MPN, including a tyrosine residue that has a possible role in catalysis and superimposes with an aspartate in related enzymes that display R selectivity towards the same substrate. Mutation of this tyrosine residue—Tyr169—in 3546-IRED to Phe resulted in a mutant of negligible activity. The data together provide structural evidence for the location and significance of the Tyr residue in this group of imine reductases, and permit a comparison of the active sites of enzymes that reduce 2MPN with either R or S selectivity.
    No preview · Article · Mar 2015 · ChemBioChem
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    ABSTRACT: The FAD-dependent monooxygenase HbpA from Pseudomonas azelaica HBP1 catalyses the hydroxylation of 2-hydroxybiphenyl (2HBP) to 2,3-dihydroxybiphenyl (23DHBP). HbpA has been used extensively as a model for studying flavoprotein hydroxylases under process conditions, and has also been subjected to directed-evolution experiments that altered its catalytic properties. The structure of HbpA has been determined in its apo and FAD-complex forms to resolutions of 2.76 and 2.03 Å, respectively. Comparisons of the HbpA structure with those of homologues, in conjunction with a model of the reaction product in the active site, reveal His48 as the most likely acid/base residue to be involved in the hydroxylation mechanism. Mutation of His48 to Ala resulted in an inactive enzyme. The structures of HbpA also provide evidence that mutants achieved by directed evolution that altered activity are comparatively remote from the substrate-binding site. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    Full-text · Article · Mar 2015 · ChemBioChem
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    ABSTRACT: Although the range of biocatalysts available for the synthesis of enantiomerically pure chiral amines continues to expand, few existing methods provide access to secondary amines. To address this shortcoming, we have over-expressed the gene for an (R)-imine reductase [(R)-IRED] from Streptomyces sp. GF3587 in Escherichia coli to create a recombinant whole-cell biocatalyst for the asymmetric reduction of prochiral imines. The (R)-IRED was screened against a panel of cyclic imines and two iminium ions and was shown to possess high catalytic activity and enantioselectivity. Preparative-scale synthesis of the alkaloid (R)-coniine (90 % yield; 99 % ee) from the imine precursor was performed on a gram-scale. A homology model of the enzyme active site, based on the structure of a closely related (R)-IRED from Streptomyces kanamyceticus, was constructed and used to identify potential amino acids as targets for mutagenesis.
    No preview · Article · Feb 2015 · ChemCatChem
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    ABSTRACT: The Baeyer-Villiger monooxygenase (BVMO) ‘MO14’ from Rhodococcus jostii RHA1, is an enantioselective BVMO that catalyses the resolution of the model ketone substrate bicyclo[3.2.0]hept-2-en-6-one to the (1S, 5R)-2-oxa lactone and the residual (1S, 5R)- substrate enantiomer. This regio- plus enantioselective behaviour is highly unusual for BVMOs, which often perform enantiodivergent biotransformations of this substrate. The scaleability of the transformation was investigated using fermentor-based experiments, in which variables including gene codon optimisation, temperature and substrate concentration were investigated. E. coli cells expressing MO14 catalysed the resolution of bicyclo[3.2.0]hept-2-en-6-one to yield (1S, 5R)-2-oxa lactone of >99% e.e and (1S, 5R)-ketone of 96% e.e. after 14 h at a temperature of 16°C and a substrate concentration of 0.5 g L-1 (4.5 mM). MO14 is thus a promising biocatalyst for the production of enantio-enriched ketones and lactones derived from the [3.2.0] platform.
    Full-text · Article · Dec 2014 · Organic & Biomolecular Chemistry
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    ABSTRACT: Flavin-containing monooxygenases (FMOs) catalyse asymmetric oxidation reactions that have potential for preparative organic synthesis, but most use the more expensive, phosphorylated nicotinamide cofactor NADPH to reduce FAD to FADH2 prior to formation of the (hydro)peroxy intermediate required for substrate oxygenation. A comparison of the structures of NADPH-dependent FMO from Methylophaga aminisulfidivorans (mFMO) and SMFMO from Stenotrophomonas maltophilia, which is able to use both NADPH and NADH, suggested that the promiscuity of the latter enzyme may be due in part to the substitution of an Arg-Thr couple in the NADPH phosphate recognition site in mFMO, for a Gln-His couple in SMFMO (Jensen et al., 2012, Chembiochem, 13, 872-878). Natural variation within the phosphate binding region, and its influence on nicotinamide cofactor promiscuity, was explored through the cloning, expression, characterisation and structural studies of FMOs from Cellvibrio sp. BR (CFMO) and Pseudomonas stutzeri NF13 (PSFMO), which possess Thr-Ser and Gln-Glu in the putative phosphate recognition positions, respectively. CFMO and PSFMO displayed 5- and 1.5-fold greater activity, respectively, than SMFMO for the reduction of FAD with NADH, and were also cofactor promiscuous, displaying a ratio of activity with NADH:NADPH of 1.7:1 and 1:1.3, respectively. The structures of CFMO and PSFMO revealed the context of the phosphate binding loop in each case, and also clarified the structure of the mobile helix-loop-helix motif that appears to shield the FAD-binding pocket from bulk solvent in this class of FMOs, a feature that was absent from the structure of SMFMO.
    Full-text · Article · Sep 2014 · Journal of Molecular Catalysis B Enzymatic
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    ABSTRACT: Alcohol dehydrogenases (ADHs) of the medium chain reductase (MDR) subfamily are valuable biocatalysts for the production of optically active alcohols and are now used routinely in industry for the preparation of synthetic intermediates. TADH from Thermus sp. ATN1 combines the advantages of both thermostability and dependence on the less expensive, non-phosphoryated, cofactor NADH as the hydride donor. The structure of TADH in complex with NADH has been determined and refined to a resolution of 2.74 A. The structure reveals structural features commensurate with known stabilising factors of thermostable MDRs, including shorter peripheral loops and an increased amount of inter- and intra-subunit salt bridges and hydrogen bonds compared to mesophilic MDRs of known structure. A study of the active site reveals the molecular determinants of NADH and substrate binding in TADH that help to shed light on observed differences in cofactor specificity and stereoselectivity when compared to its thermophilic NADPH-dependent homolog from The rmoanaerobium brockii (TbADH). The structure also provides a basis for investigating the enzyine-associated deactivation of the catalyst ([Cp*Rh(bpy)(H2O)]2(+)), which has been used in TADH-catalysed reactions for the recycling of the nicotinamide cofactor.
    Full-text · Article · Jul 2014 · Journal of Molecular Catalysis B Enzymatic
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    ABSTRACT: The (S)-selective carbonyl reductase CPCR2 from Candida parapsilosis is a member of the medium-chain reductase family of enzymes and is a useful biocatalyst for the reduction of prochiral ketone substrates. The structure of CPCR2 was determined in complex with the cofactor NADH [NADH=reduced form of nicotinamide adenine dinucleotide (NAD+)] to a resolution of 2.05 Å. Two dimers formed a tetramer in the asymmetric unit, but solution studies confirmed that a dimer was the predominant species in solution. In the monomer, the NADH cofactor is bound at the interface between the nucleotide binding domain and the catalytic domain, and the Re-face hydride of the nicotinamide ring is presented to a hydrophobic binding pocket featuring the Leu262, Phe285, Trp286, Trp116, Leu119, Leu55 and Val50 residues, which leads to the surface of the enzyme. The catalytic zinc and coordinating amino acid side chains were observed in different conformations in the different monomers. In three out of four monomers, the zinc was coordinated by His65, Asp154, Glu66 and a water molecule; in the other subunit, an alternative coordination sphere, consisting of His65, Asp154, Cys44 and a water molecule, was observed. The change in coordination was accompanied by a movement of a mobile region of the protein chain between residues 43 and 63, which bears Cys44. The structure of CPCR2 provides further evidence of a dynamic coordination sphere for zinc in medium-chain reductase dependent catalysis. It also sheds light on previous engineering studies on CPCR2 that were performed in the absence of structural data and provides a robust and reliable new model for further experiments directed towards improvement or alteration of CPCR2 activity.
    No preview · Article · Apr 2014 · ChemCatChem
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    ABSTRACT: Phloretin hydrolase from Eubacterium ramulus (Phy) catalyzes the hydrolysis of the dihydrochalcone phloretin to phloroglucinol and phloretic acid, performing a formal retro- Friedel–Crafts acylation reaction on its substrate. Its closest sequence homolog, of 25 % amino acid sequence identity, is diacetyl phloroglucinol hydrolase (Phlg) from Pseudomonas fluorescens, which catalyses a similar, hydrolytic, de-acylation of its substrate. The structure of Phlg has been determined and a catalytic mechanism proposed (J Biol Chem 285:4603–4611, 2010). In order to compare the catalytic characteristics of Phy with Phlg, the gene encoding Phy was expressed and the enzyme purified and crystallised. An X-ray fluorescence scan identified zinc within the crystals. A homology model of Phy, based on the structure of Phlg (PDB code 3HWP), informed the construction of a point mutant library of the enzyme, targeting residues shared with Phlg that are thought to be involved in zinc binding and the recognition of acyl and phenol functionality on the aromatic ring of the substrates. Mutation of His123, His251, Glu154 and Glu255 (conserved zinc binding residues) resulted in variants that were either poorly expressed, or of much reduced activity; Mutation of Tyr115 and His203, thought to bind the phenol groups in the 1-and 3-positions of the phloroglucinol ring respectively, resulted in variants of 15-fold reduced activity and an inactive variant. These results are suggestive of conservation of some aspects of mechanism and substrate recognition between Phy and Phlg, and of the catalytic characteristics of Zn-dependent C–C hydrolases of this type in general.
    No preview · Article · Mar 2014 · Topics in Catalysis
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    ABSTRACT: Alcohol dehydrogenases (ADHs) are applied in industrial synthetic chemistry for the production of optically active secondary alcohols. However, the substrate spectrum of many ADHs is narrow, and few, for example, are suitable for the reduction of prochiral ketones in which the carbonyl group is bounded by two bulky and/or hydrophobic groups; so-called ‘bulky–bulky’ ketones. Recently two ADHs, RasADH from Ralstonia sp. DSM 6428, and SyADH from Sphingobium yanoikuyae DSM 6900, have been described, which are distinguished by their ability to accept bulky–bulky ketones as substrates. In order to examine the molecular basis of the recognition of these substrates the structures of the native and NADPH complex of RasADH, and the NADPH complex of SyADH have been determined and refined to resolutions of 1.5, 2.9 and 2.5 Å, respectively. The structures reveal hydrophobic active site tunnels near the surface of the enzymes that are well-suited to the recognition of large hydrophobic substrates, as determined by modelling of the bulky–bulky substrate n-pentyl phenyl ketone. The structures also reveal the bases for NADPH specificity and (S)-stereoselectivity in each of the biocatalysts for n-pentyl phenyl ketone and related substrates.
    No preview · Article · Mar 2014 · Topics in Catalysis
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    ABSTRACT: The flavoprotein monooxygenase (FPMO) from Stenotrophomonas maltophilia (SMFMO, Uniprot: B2FLR2) catalyses the asymmetric oxidation of thioethers and is unusual amongst FPMOs in its ability to use the non-phosphorylated cofactor NADH, as well as NADPH, for the reduction of the FAD coenzyme. In order to explore the basis for cofactor promiscuity, structure-guided mutation of two residues in the cofactor binding site, Gln193 and His194, in SMFMO were performed in an attempt to imitate the cofactor binding site of the NADPH-dependent FMO from Methylophaga aminisulfidivorans sp. SK1 (mFMO), in which structurally homologous residues Arg234 and Thr235 bind the NADPH 2'-ribose phosphate. Mutation of His194 to threonine proved most significant, with a switch in specificity from NADH to NADPH [(k cat/K m NADH)/k cat/K m NADPH) from 1.5:1 to 1:3.5, mostly as a result of a reduced K m for NADPH of approximately sevenfold in the His194Thr mutant. The structure of the Gln193Arg/His194Thr mutant revealed no substantial changes in the backbone of the enzyme or orientation of side chains resulting from mutation. Mutation of Phe52, in the vicinity of FAD, and which in mFMO is an asparagine thought to be responsible for flavin hydroperoxide stabilisation, is, in SMFMO, a determinant of enantioselectivity in sulfoxidation. Mutation of Phe52 to valine resulted in a mutant that transformed para-tolyl methyl sulfide into the (S)-sulfoxide with 32% e.e., compared to 25% (R)- for the wild type. These results shed further light both on the cofactor specificity of FPMOs, and their determinants of enantioselectivity, with a view to informing engineering studies of FPMOs in the future.
    Full-text · Article · Dec 2013 · FEBS Open Bio
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    ABSTRACT: Baeyer–Villiger monooxygenases (BVMOs) are valuable enzymes for specific oxyfunctionalization chemistry. They catalyze the oxidation of ketones to esters, but are also capable of oxidizing other chemical functions, namely aldehydes and heteroatoms such as sulfur, nitrogen, selenium and boron. The oxidation specificity and enantioselectivity of a newly characterized BVMO (BVMO4) from a strain of Dietzia towards sulfide- and aldehyde substrates have been studied. BVMO4 could react with sulfides containing an aromatic group. The presence of a substituent on the aromatic group was tolerated when they were in the meta- and para position and the oxidations yielded predominantly the (R)-sulfoxides. Similarly, BVMO4 displayed a higher activity for aldehydes containing a phenyl group, but long aliphatic aldehydes, namely octanal and decanal, were also accepted as substrate by this enzyme. The major oxidation products of the aldehyde substrates were the respective carboxylic acids in contrast to formate ester that was obtained in most of the previous reports. The Baeyer–Villiger oxidation of the substrate 2-phenylpropionaldehyde was studied in further detail and the corresponding acid product was obtained with good regio- and enantioselectivity. This is a unique feature for BVMO4 and is of great interest for further exploration of an alternative biocatalytic process.
    Full-text · Article · Nov 2013 · Topics in Catalysis

  • No preview · Article · Sep 2013 · ChemInform
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    ABSTRACT: The ol' switcheroo: The enantioselectivity of an O2 -dependent hydroxylation at a mononuclear non-heme iron center was switched from S to R by changing the geometry of the substrate ligand at the metal center through protein redesign. This rational approach resulted in a greater than 9000-fold enhancement in (R)-selectivity and yielded a highly active (R)-mandelate synthase.
    No preview · Article · Sep 2013 · Angewandte Chemie International Edition
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    ABSTRACT: NADPH-dependent oxidoreductase Q1EQE0 from Streptomyces kanamyceticus catalyzes the asymmetric reduction of the prochiral monocyclic imine 2-methyl-1-pyrroline to the chiral amine (R)-2-methylpyrrolidine with >99 % ee, and is thus of interest as a potential biocatalyst for the production of optically active amines. The structures of Q1EQE0 in native form, and in complex with the nicotinamide cofactor NADPH have been solved and refined to a resolution of 2.7 Å. Q1EQE0 functions as a dimer in which the monomer consists of an N-terminal Rossman-fold motif attached to a helical C-terminal domain through a helix of 28 amino acids. The dimer is formed through reciprocal domain sharing in which the C-terminal domains are swapped, with a substrate-binding cleft formed between the N-terminal subunit of monomer A and the C-terminal subunit of monomer B. The structure is related to those of known β-hydroxyacid dehydrogenases, except that the essential lysine, which serves as an acid/base in the (de)protonation of the nascent alcohol in those enzymes, is replaced by an aspartate residue, Asp187 in Q1EQE0. Mutation of Asp187 to either asparagine or alanine resulted in an inactive enzyme.
    No preview · Article · Jul 2013 · ChemBioChem
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    ABSTRACT: The development of cost-effective and sustainable catalytic methods for the production of enantiomerically pure chiral amines is a key challenge facing the pharmaceutical and fine chemical industries. This challenge is highlighted by the estimate that 40-45% of drug candidates contain a chiral amine, fuelling a demand for broadly applicable synthetic methods which deliver target structures in high yield and enantiomeric excess (e.e.). Herein we describe the development and application of a 'toolbox' of monoamine oxidase variants from Aspergillus niger (MAO-N) which display remarkable substrate scope and tolerance for sterically demanding motifs, including a new variant which exhibits high activity and enantioselectivity towards substrates containing the aminodiphenylmethane (benzhydrylamine) template. By combining rational structure-guided engineering with high-throughput screening, it has been possible to expand the substrate scope of MAO-N to accommodate amine substrates containing bulky aryl substituents. These engineered MAO-N biocatalysts have been applied in deracemisation reactions for the efficient asymmetric synthesis of the generic APIs Solifenacin and Levocetirizine as well as the natural products (R)-coniine, (R)-eleagnine and (R)-leptaflorine. We also report a novel MAO-N mediated asymmetric oxidative Pictet-Spengler approach to the synthesis of (R)-harmicine.
    No preview · Article · Jun 2013 · Journal of the American Chemical Society

Publication Stats

2k Citations
361.49 Total Impact Points

Institutions

  • 2001-2015
    • The University of York
      • • Department of Chemistry
      • • York Structural Biology Laboratory
      York, England, United Kingdom
  • 2004-2014
    • CUNY Graduate Center
      New York, New York, United States
  • 2012
    • The University of Manchester
      • School of Chemistry
      Manchester, England, United Kingdom
  • 2010
    • University of Wales
      • Department of Chemistry
      Cardiff, Wales, United Kingdom
  • 1998-2010
    • The University of Edinburgh
      • School of Chemistry
      Edinburgh, Scotland, United Kingdom
  • 1992-2010
    • University of Exeter
      Exeter, England, United Kingdom
  • 2008
    • Institute of Food Research
      Norwich, England, United Kingdom
  • 2006
    • Vienna University of Technology
      • Institute of Applied Synthetic Chemistry
      Wien, Vienna, Austria