GDP-4-KETO-6-DEOXY-D-MANNOSE EPIMERASE REDUCTASE FROM ESCHERICHIA-COLI, A KEY ENZYME IN THE BIOSYNTHESIS OF GDP-L-FUCOSE, DISPLAYS THE STRUCTURAL CHARACTERISTICS OF THE RED PROTEIN HOMOLOGY SUPERFAMILY/

Background: The process of guanosine 5'-diphosphate L-fucose (GDP-L-fu cose) biosynthesis is conserved throughout evolution from prokaryotes to man. In animals, GDP-L-fucose is the substrate of fucosyltransferas es that participate in the biosynthesis and remodeling of glycoconjuga tes, including ABH blood group and Lewis-system antigens. The 'de novo ' pathway of GDP-L-fucose biosynthesis from GDP-D-mannose involves a G DP-D-mannose 4,6 dehydratase (GMD) and a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase (GMER). Neither of the catalytic mechanisms nor th e three-dimensional structures of the two enzymes has been elucidated yet. The severe leukocyte adhesion deficiency (LAD) type II genetic sy ndrome is known to result from deficiencies in this de novo pathway. R esults: The crystal structures of apo- and holo-GMER have been determi ned at 2.1 Angstrom and 2.2 Angstrom resolution, respectively, Each su bunit of the homodimeric (2 x 34 kDa) enzyme is composed of two domain s. The N-terminal domain, a six-stranded Rossmann fold, binds NADP(+); the C-terminal domain (about 100 residues) displays an alpha/beta top ology. NADP(+) interacts with residues Arg12 and Arg36 at the adenylic ribose phosphate; moreover, a protein loop based on the Gly-X-X-Gly-X -X-Gly motif (where X is any amino acid) stabilizes binding of the coe nzyme diphosphate bridge. The nicotinamide and the connected ribose ri ng are located close to residues Ser107, Tyr136 and Lys140, the putati ve GMER active-site center. Conclusions: The GMER fold is reminiscent of that observed for UDP-galactose epimerase (UGE) from Escherichia co li. Consideration of the enzyme fold and of its main structural featur es allows assignment of GMER to the reductase-epimerase-dehydrogenase (RED) enzyme homology superfamily, to which short-chain dehydrogenase/ reductases (SDRs) also belong. The location of the NADP(+) nicotinamid e ring at an interdomain cleft is compatible with; substrate binding i n the C-terminal domain.