Probing the catalytic mechanism of GDP-4-keto-6-deoxy-D-mannose epimerase/reductase by kinetic and crystallographic characterization of site-specificmutants

GDP-4-keto-6-deoxy-D-mannose epimerase/reductase is a bifunctional enzyme r esponsible for the last step in the biosynthesis of GDP-(L)-fucose, the sub strate of fucosyl transferases. Several cell-surface antigens, including th e leukocyte Lewis system and cell-surface antigens in pathogenic bacteria, depend on the availability of GDP-(L)-fucose for their expression. Therefor e, the enzyme is a potential target for therapy in pathological states depe nding on selectin-mediated cell-to-cell interactions. Previous crystallogra phic investigations have shown that GDP-4-keto-6-deoxy-D-mannose epimerase/ reductase belongs to the short-chain dehydrogenase/reductase protein homolo gy family. The enzyme active-site region is at the interface of an N-termin al NADPH-binding domain and a C-terminal domain, held to bind the substrate . The design, expression and functional characterization of seven site-spec ific mutant forms of GDP-4-keto-6-deoxy-D-mannose epimerase/reductase are r eported here. In parallel, the crystal structures of the native holoenzyme and of three mutants (Ser107Ala, Tyr136Glu and Lys140Arg) have been investi gated and refined at 1.45-1.60 Angstrom resolution, based on synchrotron da ta (X-factors range between 12.6% and 13.9 %). The refined protein models s how that besides the active-site residues Ser107, Tyr136 and Lys140, whose mutations impair the overall enzymatic activity and may affect the coenzyme binding mode, side-chains capable of proton exchange, located around the e xpected substrate (GDP-4-keto-6-deoxy-D-mannose) binding pocket, are select ively required during the epimerization and reduction steps. Among these, C ys109 and His179 may play a primary role in proton exchange between the enz yme and the epimerization catalytic intermediates. Finally, the additional role of mutated active-site residues involved in substrate recognition and in enzyme stability has been analyzed. (C) 2000 Academic Press.