Evidence that NADP+ is the physiological cofactor of ADP-L-glycero-D-mannoheptose 6-epimerase.
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 microm whereas that for NAD(+) was 45 microm. 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(+).
Ni
Y
Y
Laboratory of Biochemistry and Genetics, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA.
McPhie
P
P
Deacon
A
A
Ealick
S
S
Coleman
W G
WG
D000227:Adenine Nucleotides / Q000378:metabolism*; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009249:NADP / Q000378:metabolism*; D011487:Protein Conformation
D000227:Adenine Nucleotides; D009249:NADP; C037213:ADP-L-glycero-D-mannoheptose-6-epimerase; D002238:Carbohydrate Epimerases