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J. Biol. Chem., Vol. 276, Issue 29, 27329-27334, July 20, 2001

This Article Full Text Full Text (PDF) All Versions of this Article: 276/29/27329    most recent M102258200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ni, Y. Articles by Coleman, W. G. Search for Related Content PubMed PubMed Citation Articles by Ni, Y. Articles by Coleman, W. G., Jr. Social Bookmarking                      What's this?

Evidence that NADP⁺ Is the Physiological Cofactor of ADP-L-glycero-D-mannoheptose 6-Epimerase^*

YiSheng Ni, Peter McPhie, Ashley Deacon^§, Steve Ealick^¶, and William G. Coleman Jr.

From the  Laboratory of Biochemistry and Genetics, NIDDK, National Institutes of Health, Bethesda, MD 20892, ^§ Stanford Linear Accelerator Center, Stanford University, Stanford, CA 94309, and ^¶ Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853

ADP-L-glycero-D-mannoheptose 6-epimerase is required for lipopolysaccharide inner core biosynthesis in several genera of Gram-negative bacteria. The enzyme contains both fingerprint sequences Gly-X-Gly-X-X-Gly and Gly-X-X-Gly-X-X-Gly near its N terminus, which is indicative of an ADP binding fold. Previous studies of this ADP-L-glycero-D-mannoheptose 6-epimerase (ADP-hep 6-epimerase) were consistent with an NAD⁺ cofactor. However, the crystal structure of this ADP-hep 6-epimerase showed bound NADP (Deacon, A. M., Ni, Y. S., Coleman, W. G., Jr., and Ealick, S. E. (2000) Structure 5, 453-462). In present studies, apo-ADP-hep 6-epimerase was reconstituted with NAD⁺, NADP⁺, and FAD. In this report we provide data that shows NAD⁺ and NADP⁺ both restored enzymatic activity, but FAD could not. Furthermore, ADP-hep 6-epimerase exhibited a preference for binding of NADP⁺ over NAD⁺. The K_d value for NADP⁺ was 26 µM whereas that for NAD⁺ was 45 µM. Ultraviolet circular dichroism spectra showed that apo-ADP-hep 6-epimerase reconstituted with NADP⁺ had more secondary structure than apo-ADP-hep 6-epimerase reconstituted with NAD⁺. Perchloric acid extracts of the purified enzyme were assayed with NAD⁺-specific alcohol dehydrogenase and NADP⁺-specific isocitric dehydrogenase. A sample of the same perchloric acid extract was analyzed in chromatographic studies, which demonstrated that ADP-hep 6-epimerase binds NADP⁺ in vivo. A structural comparison of ADP-hep 6-epimerase with UDP-galactose 4-epimerase, which utilizes an NAD⁺ cofactor, has identified the regions of ADP-hep 6-epimerase, which defines its specificity for NADP⁺.

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