The mechanism of the reaction catalyzed by ADP-beta-L-glycero-D-manno-heptose 6-epimerase.
ADP-l-glycero-d-manno-heptose 6-epimerase (AGME, RfaD) is a bacterial enzyme that is involved in lipopolysaccharide biosynthesis and interconverts ADP-beta-l-glycero-d-manno-heptose (ADP-l,d-Hep) with ADP-beta-d-glycero-d-manno-heptose (ADP-d,d-Hep). AGME is known to require a tightly bound NADP+ cofactor for activity and presumably employs a mechanism involving transient oxidation of the substrate. Four mechanistic possibilities are considered that involve transient oxidation at either C-7' ', C-6' ', or C-4' ' of the heptose nucleotide. In this contribution, the use of solvent isotope incorporation studies and alternate substrates provides strong evidence for a mechanism involving nonstereospecific oxidation/reduction directly at C-6' '. It was found that the epimerization proceeds without any detectable incorporation of solvent-derived deuterium or 18O-isotope into the product. This argues against mechanisms involving either proton transfers at carbon or dehydration/rehydration events. In addition, the deoxygenated analogues, 7' '-deoxy-ADP-l,d-Hep and 4' '-deoxy-ADP-l,d-Hep, were both found to serve as substrates for the enzyme, indicating that oxidation at either C-7' ' or C-4' ' is not required for catalysis.
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Jay A
JA
Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada.
Ahmed
Raef A
RA
Morrison
James P
JP
Coleman
William G
WG
Tanner
Martin E
ME
D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006539:Heptoses / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D006539:Heptoses; D011994:Recombinant Proteins; C062615:glycero-alpha-manno-heptopyranose; C037213:ADP-L-glycero-D-mannoheptose-6-epimerase; D002238:Carbohydrate Epimerases