title: Structure and biocatalytic scope of thermophilic flavin-dependent halogenase and flavin reductase enzymes.
abstract: Flavin-dependent halogenase (Fl-Hal) enzymes have been shown to halogenate a range of synthetic as well as natural aromatic compounds. The exquisite regioselectively of Fl-Hal enzymes can provide halogenated building blocks which are inaccessible using standard halogenation chemistries. Consequently, Fl-Hal are potentially useful biocatalysts for the chemoenzymatic synthesis of pharmaceuticals and other valuable products, which are derived from haloaromatic precursors. However, the application of Fl-Hal enzymes, in vitro, has been hampered by their poor catalytic activity and lack of stability. To overcome these issues, we identified a thermophilic tryptophan halogenase (Th-Hal), which has significantly improved catalytic activity and stability, compared with other Fl-Hal characterised to date. When used in combination with a thermostable flavin reductase, Th-Hal can efficiently halogenate a number of aromatic substrates. X-ray crystal structures of Th-Hal, and the reductase partner (Th-Fre), provide insights into the factors that contribute to enzyme stability, which could guide the discovery and engineering of more robust and productive halogenase biocatalysts.
authors.affiliation: School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK. jason.micklefield@manchester.ac.uk.
authors.forename: Binuraj R K
authors.initials: BR
authors.lastname: Menon
authors.affiliation: School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK. jason.micklefield@manchester.ac.uk.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Latham
authors.affiliation: School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK. jason.micklefield@manchester.ac.uk.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Dunstan
authors.affiliation: School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK. jason.micklefield@manchester.ac.uk.
authors.forename: Eileen
authors.initials: E
authors.lastname: Brandenburger
authors.affiliation: School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK. jason.micklefield@manchester.ac.uk.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Klemstein
authors.affiliation: School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK. jason.micklefield@manchester.ac.uk.
authors.forename: David
authors.initials: D
authors.lastname: Leys
authors.affiliation: School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK. jason.micklefield@manchester.ac.uk.
authors.forename: Chinnan
authors.initials: C
authors.lastname: Karthikeyan
authors.affiliation: School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK. jason.micklefield@manchester.ac.uk.
authors.forename: Michael F
authors.initials: MF
authors.lastname: Greaney
authors.affiliation: School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK. jason.micklefield@manchester.ac.uk.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Shepherd
authors.affiliation: School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK. jason.micklefield@manchester.ac.uk.
authors.forename: Jason
authors.initials: J
authors.lastname: Micklefield
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D038181:FMN Reductase / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology; D013379:Substrate Specificity; D044366:Transition Temperature
chemical_list: D001426:Bacterial Proteins; D010088:Oxidoreductases; C407363:tryptophan halogenase; D038181:FMN Reductase