Structural and kinetic analysis of Escherichia coli GDP-mannose 4,6 dehydratase provides insights into the enzyme's catalytic mechanism and regulation by GDP-fucose.
BACKGROUND
GDP-mannose 4,6 dehydratase (GMD) catalyzes the conversion of GDP-(D)-mannose to GDP-4-keto, 6-deoxy-(D)-mannose. This is the first and regulatory step in the de novo biosynthesis of GDP-(L)-fucose. Fucose forms part of a number of glycoconjugates, including the ABO blood groups and the selectin ligand sialyl Lewis X. Defects in GDP-fucose metabolism have been linked to leukocyte adhesion deficiency type II (LADII).


RESULTS
The structure of the GDP-mannose 4,6 dehydratase apo enzyme has been determined and refined using data to 2.3 A resolution. GMD is a homodimeric protein with each monomer composed of two domains. The larger N-terminal domain binds the NADP(H) cofactor in a classical Rossmann fold and the C-terminal domain harbors the sugar-nucleotide binding site. We have determined the GMD dissociation constants for NADP, NADPH and GDP-mannose. Each GMD monomer binds one cofactor and one substrate molecule, suggesting that both subunits are catalytically competent. GDP-fucose acts as a competitive inhibitor, suggesting that it binds to the same site as GDP-mannose, providing a mechanism for the feedback inhibition of fucose biosynthesis.


CONCLUSIONS
The X-ray structure of GMD reveals that it is a member of the short-chain dehydrogenase/reductase (SDR) family of proteins. We have modeled the binding of NADP and GDP-mannose to the enzyme and mutated four of the active-site residues to determine their function. The combined modeling and mutagenesis data suggests that at position 133 threonine substitutes serine as part of the serine-tyrosine-lysine catalytic triad common to the SDR family and Glu 135 functions as an active-site base.
Somoza
J R
JR
Wyeth Research, Cambridge, MA 02140, USA.
Menon
S
S
Schmidt
H
H
Joseph-McCarthy
D
D
Dessen
A
A
Stahl
M L
ML
Somers
W S
WS
Sullivan
F X
FX
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D020134:Catalytic Domain; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology*; D006154:Guanosine Diphosphate Fucose / Q000378:metabolism*; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D006154:Guanosine Diphosphate Fucose; D009249:NADP; D006836:Hydro-Lyases; C044971:GDPmannose 4,6-dehydratase