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Instruments / Technical Area	Publication Details	Published	Actions
I04-Macromolecular Crystallography	A reductive aminase switches to imine reductase mode for a bulky amine substrate Amelia K. Gilio , Thomas W. Thorpe , Alex Heyam , Mark R. Petchey , Balázs Pogrányi , Scott P. France , Roger M. Howard , Michael J. Karmilowicz , Russell Lewis , Nicholas Turner , Gideon Grogan Acs Catalysis DOI: 10.1021/acscatal.2c06066 Diamond Proposal Number(s): [18598] Open Access Show Abstract ▼ Abstract: Imine reductases (IREDs) catalyze the asymmetric reduction of cyclic imines, but also in some cases the coupling of ketones and amines to form secondary amine products in an enzyme-catalyzed reductive amination (RedAm) reaction. Enzymatic RedAm reactions have typically used small hydrophobic amines, but many interesting pharmaceutical targets require that larger amines be used in these coupling reactions. Following the identification of IR77 from Ensifer adhaerens as a promising biocatalyst for the reductive amination of cyclohexanone with pyrrolidine, we have characterized the ability of this enzyme to catalyze couplings with larger bicyclic amines such as isoindoline and octahydrocyclopenta(c)pyrrole. By comparing the activity of IR77 with reductions using sodium cyanoborohydride in water, it was shown that, while the coupling of cyclohexanone and pyrrolidine involved at least some element of reductive amination, the amination with the larger amines likely occurred ex situ, with the imine recruited from solution for enzyme reduction. The structure of IR77 was determined, and using this as a basis, structure-guided mutagenesis, coupled with point mutations selecting improving amino acid sites suggested by other groups, permitted the identification of a mutant A208N with improved activity for amine product formation. Improvements in conversion were attributed to greater enzyme stability as revealed by X-ray crystallography and nano differential scanning fluorimetry. The mutant IR77-A208N was applied to the preparative scale amination of cyclohexanone at 50 mM concentration, with 1.2 equiv of three larger amines, in isolated yields of up to 93%.	Jan 2023
I04-1-Macromolecular Crystallography (fixed wavelength)	Profiling substrate promiscuity of wild-type sugar kinases for multi-fluorinated monosaccharides Tessa Keenan , Fabio Parmeggiani , Julien Malassis , Clement Q. Fontenelle , Jean-Baptiste Vendeville , Wendy Offen , Peter Both , Kun Huang , Andrea Marchesi , Alex Heyam , Carl Young , Simon J. Charnock , Gideon J. Davies , Bruno Linclau , Sabine L. Flitsch , Martin A. Fascione Cell Chemical Biology DOI: 10.1016/j.chembiol.2020.06.005 Diamond Proposal Number(s): [13587, 18598] Show Abstract ▼ Abstract: Fluorinated sugar-1-phosphates are of emerging importance as intermediates in the chemical and biocatalytic synthesis of modified oligosaccharides, as well as probes for chemical biology. Here we present a systematic study of the activity of a wide range of anomeric sugar kinases (galacto- and N-acetylhexosamine kinases) against a panel of fluorinated monosaccharides, leading to the first examples of polyfluorinated substrates accepted by this class of enzymes. We have discovered four new N-acetylhexosamine kinases with a different substrate scope, thus expanding the number of homologs available in this subclass of kinases. Lastly, we have solved the crystal structure of a galactokinase in complex with 2-deoxy-2-fluorogalactose, giving insight into changes in the active site that may account for the specificity of the enzyme toward certain substrate analogs.	Jul 2020

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