title: Dismutase activity of ADP-L-glycero-D-manno-heptose 6-epimerase: evidence for a direct oxidation/reduction mechanism.
abstract: The first positive evidence for the utilization of a direct C-6' ' oxidation/reduction mechanism by ADP-l-glycero-d-manno-heptose 6-epimerase is reported here. The epimerase (HldD or AGME, formerly RfaD) operates in the biosynthetic pathway of l-glycero-d-manno-heptose, which is a conserved sugar in the core region of lipopolysaccharide (LPS) of Gram-negative bacteria. The stereochemical inversion catalyzed by the epimerase is interesting as it occurs at an "unactivated" stereocenter that lacks an acidic C-H bond, and therefore, a direct deprotonation/reprotonation mechanism cannot be employed. Instead, the epimerase employs a transient oxidation strategy involving a tightly bound NADP(+) cofactor. A recent study ruled out mechanisms involving transient oxidation at C-4' ' and C-7' ' and supported a mechanism that involves an initial oxidation directly at the C-6' ' position to generate a 6' '-keto intermediate (Read, J. A., Ahmed, R. A., Morrison, J. P., Coleman, W. G., Jr., Tanner, M. E. (2004) J. Am. Chem. Soc. 126, 8878-8879). A subsequent nonstereospecific reduction of the ketone intermediate can generate either epimer of the ADP-heptose. In this work, an intermediate analogue containing an aldehyde functionality at C-6' ', ADP-beta-d-manno-hexodialdose, is prepared in order to probe the ability of the enzyme to catalyze redox chemistry at this position. It is found that incubation of the aldehyde with a catalytic amount of the epimerase leads to a dismutation process in which one-half of the material is oxidized to ADP-beta-d-mannuronic acid and the other half is reduced to ADP-beta-d-mannose. Transient reduction of the enzyme-bound NADP(+) was monitored by UV spectroscopy and implicates the cofactor's involvement during catalysis.
authors.affiliation: Department of Chemistry, University of British Columbia, Vancouver, British Columbia, V6T 1Z1, Canada.
authors.forename: James P
authors.initials: JP
authors.lastname: Morrison
authors.forename: Jay A
authors.initials: JA
authors.lastname: Read
authors.forename: William G
authors.initials: WG
authors.lastname: Coleman
authors.forename: Martin E
authors.initials: ME
authors.lastname: Tanner
mesh_terms: D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008954:Models, Biological; D009249:NADP / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D004269:DNA, Bacterial; D011994:Recombinant Proteins; D009249:NADP; C037213:ADP-L-glycero-D-mannoheptose-6-epimerase; D002238:Carbohydrate Epimerases