|Title||Dismutase activity of ADP-L-glycero-D-manno-heptose 6-epimerase: Evidence for a direct oxidation/reduction mechanism|
|Publication Type||Journal Article|
|Year of Publication||2005|
|Authors||Morrison, JP, Read, JA, Coleman, WG, Tanner, ME|
|Type of Article||Article|
|Keywords||ACID, ALCOHOL-DEHYDROGENASE, ALDEHYDE DEHYDROGENASE, D-MANNOHEPTOSE 6-EPIMERASE, DROSOPHILA-MELANOGASTER, EPIMERIZATION, ESCHERICHIA-COLI, GLUCOSE DEHYDROGENASE, LIVER, SITE RESIDUES, UDP-GALACTOSE 4-EPIMERASE|
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.
|URL||<Go to ISI>://000228425600037|