PMID:15823050

Citation	Morrison, JP, Read, JA, Coleman, WG Jr and Tanner, ME (2005) Dismutase activity of ADP-L-glycero-D-manno-heptose 6-epimerase: evidence for a direct oxidation/reduction mechanism. Biochemistry 44:5907-15
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.
Links	PubMed Online version:10.1021/bi050106c
Keywords	Base Sequence; Carbohydrate Epimerases/chemistry; Carbohydrate Epimerases/genetics; Carbohydrate Epimerases/metabolism; Cloning, Molecular; DNA, Bacterial/genetics; Escherichia coli/enzymology; Escherichia coli/genetics; Models, Biological; NADP/metabolism; Nuclear Magnetic Resonance, Biomolecular; Oxidation-Reduction; Recombinant Proteins/chemistry; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Spectrometry, Mass, Electrospray Ionization; Stereoisomerism; Substrate Specificity
edit table

Significance

You can help EcoliWiki by summarizing why this paper is useful

Useful Materials and Methods

You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.

Annotations

<annotationlinks/>

EcoliWiki Links

Add links to pages that link here (e.g. gene, product, method pages)

References

See Help:References for how to manage references in EcoliWiki.