COFACTOR CHARACTERIZATION AND MECHANISTIC STUDIES OF CDP-6-DEOXY-DELTA(3,4)-GLUCOSEEN REDUCTASE - EXPLORATION INTO A NOVEL ENZYMATIC C-O BOND-CLEAVAGE EVENT

The CDP-6-deoxy-DELTA3,4-glucoseen reductase (E3) is a NADH-dependent enzyme which catalyzes the key reduction of the C-3 deoxygenation step during the formation of CDP-ascarylose, a 3,6-dideoxyhexose found in the lipopolysaccharide of Yersinia pseudotuberculosis. This highly pur ified enzyme is also a NADH oxidase capable of mediating the direct el ectron transfer from NADH to O2, forming H2O2. While previous work sho wed that E3 contains no common cofactor, one FAD and one plant ferredo xin type [2Fe-2S] center were found in this study to be associated wit h each molecule of E3. The iron-sulfur center is essential for E3 acti vity since bleaching of the [2Fe-2S] center leads to inactive enzyme. These results suggest that E3 employs a short electron-transport chain composed of both FAD and the iron-sulfur center to shuttle electrons from NADH to its acceptor. The order of electron flow, as indicated by EPR measurement with partially reduced E3, starts with hydride reduct ion of FAD by NADH. The iron-sulfur cluster, receiving electrons one a t a time from the reduced flavin, relays the reducing equivalents via another iron-sulfur center in the active site of E1 to its final accep tor, the E1-bound PMP-glucoseen adduct. The participation of a one-ele ctron-carrying iron-sulfur center in this reduction is advantageous si nce both electrons are dispatched from the same redox state of the pro sthetic group, allowing electrons of equal energy to be delivered to t he final acceptor. This proposed electron-transport sequence is consis tent with the role of E3 as a 2e-/1e- switch and provides compelling e vidence supporting a radical mechanism for E3-catalyzed C-3 deoxygenat ion. In light of the fact that a PMP-glucoseen adduct is the ultimate acceptor receiving electrons from E3, the catalytic role of E3 in the biosynthesis of ascarylose clearly constitutes a unique example of bio logical deoxygenation.