title: Evaluation of fluoropyruvate as nucleophile in reactions catalysed by N-acetyl neuraminic acid lyase variants: scope, limitations and stereoselectivity.
abstract: The catalysis of reactions involving fluoropyruvate as donor by N-acetyl neuraminic acid lyase (NAL) variants was investigated. Under kinetic control, the wild-type enzyme catalysed the reaction between fluoropyruvate and N-acetyl mannosamine to give a 90 : 10 ratio of the (3R,4R)- and (3S,4R)-configured products; after extended reaction times, equilibration occurred to give a 30 : 70 mixture of these products. The efficiency and stereoselectivity of reactions of a range of substrates catalysed by the E192N, E192N/T167V/S208V and E192N/T167G NAL variants were also studied. Using fluoropyruvate and (2R,3S)- or (2S,3R)-2,3-dihydroxy-4-oxo-N,N-dipropylbutanamide as substrates, it was possible to obtain three of the four possible diastereomeric products; for each product, the ratio of anomeric and pyranose/furanose forms was determined. The crystal structure of S. aureus NAL in complex with fluoropyruvate was determined, assisting rationalisation of the stereochemical outcome of C-C bond formation.
authors.affiliation: School of Chemistry, University of Leeds, Leeds, LS2 9JT, UK. a.s.nelson@leeds.ac.uk.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Stockwell
authors.forename: Adam D
authors.initials: AD
authors.lastname: Daniels
authors.forename: Claire L
authors.initials: CL
authors.lastname: Windle
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Harman
authors.forename: Thomas
authors.initials: T
authors.lastname: Woodhall
authors.forename: Tomas
authors.initials: T
authors.lastname: Lebl
authors.forename: Chi H
authors.initials: CH
authors.lastname: Trinh
authors.forename: Keith
authors.initials: K
authors.lastname: Mulholland
authors.forename: Arwen R
authors.initials: AR
authors.lastname: Pearson
authors.forename: Alan
authors.initials: A
authors.lastname: Berry
authors.forename: Adam
authors.initials: A
authors.lastname: Nelson
mesh_terms: D055162:Biocatalysis; D050113:Imino Furanoses / Q000737:chemistry / Q000378:metabolism*; D050112:Imino Pyranoses / Q000737:chemistry / Q000378:metabolism*; D008968:Molecular Conformation; D007652:Oxo-Acid-Lyases / Q000378:metabolism*; D011773:Pyruvates / Q000737:chemistry / Q000378:metabolism*; D013237:Stereoisomerism
chemical_list: D050113:Imino Furanoses; D050112:Imino Pyranoses; D011773:Pyruvates; C000610682:fluoropyruvate; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase