|Title||Intermediate release by ADP-L-glycero-D-manno-heptose 6-epimerase|
|Publication Type||Journal Article|
|Year of Publication||2007|
|Authors||Mayer, A, Tanner, ME|
ADP-L-glycero-D-manno-heptose 6-epimerase (HldD or AGME, formerly RfaD) catalyzes the interconversion of ADP-beta-D-glycero-D-manno-heptose (ADP-D,D-Hep) and ADP-beta-L-glycero-D-manno-heptose (ADP-L, D-Hep). The latter compound provides the heptose moiety that is used in lipopolysaccharide biosynthesis by Gram-negative bacteria. Several lines of evidence suggest that the enzyme uses a direct oxidation/reduction mechanism involving a tightly bound NADP(+) cofactor. An initial oxidation at C-6" gives a 6"-keto intermediate, and a subsequent reduction on the opposite face of the carbonyl group generates the epimeric product. The reorientation required for the nonstereoselective reduction could take place within a single active site, or it could involve the release of the intermediate and rebinding in an altered conformation. To distinguish between these possibilities, two isotopically labeled substrates (ADPD, D-Hep) were prepared that contained O-18 and H-2 isotopes at C-7" and C-6", respectively. A crossover experiment was used to determine whether unlabeled or doubly labeled products were formed upon epimerization of a mixture of the two singly labeled compounds. After an initial epimeric equilibrium was reached, no crossover could be detected, indicating that intermediate release is not intrinsic to the overall mechanism. After extended incubation, however, scrambling of the labels could be detected, indicating that a low background rate of intermediate release does occur. To directly detect the release of the intermediate, the labeled compounds were independently epimerized in the presence of a ketone-trapping reagent, phenylhydrazine. The corresponding phenylhydrazones were identified by mass spectrometry, and the absence of any 2H isotope in the adduct obtained from the deuterated starting compound confirmed that the oxidation had occurred at C-6".
|URL||<Go to ISI>://000246456200029|