TY - JOUR
T1 - Cofactor Characterization and Mechanistic Studies of CDP-6-deoxy-Δ3,4-glucoseen Reductase
T2 - Exploration into a Novel Enzymatic C-O Bond Cleavage Event
AU - Miller, Vaughn P.
AU - Thorson, Jon S.
AU - Ploux, Olivier
AU - Lo, Stanley F.
AU - Liu, Hung Wen
PY - 1993
Y1 - 1993
N2 - The CDP-6-deoxy-Δ3,4-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 purified enzyme is also a NADH oxidase capable of mediating the direct electron transfer from NADH to O2, forming H2O2. While previous work showed that E3 contains no common cofactor, one FAD and one plant ferredoxin type [2Fe-2S] center were found in this study to be associated with each molecule of E3. The iron-sulfur center is essential for E3 activity 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 reduction of FAD by NADH. The iron-sulfur cluster, receiving electrons one at a time from the reduced flavin, relays the reducing equivalents via another iron-sulfur center in the active site of E1 to its final acceptor, the E1-bound PMP-glucoseen adduct. The participation of a one-electron-carrying iron-sulfur center in this reduction is advantageous since both electrons are dispatched from the same redox state of the prosthetic group, allowing electrons of equal energy to be delivered to the final acceptor. This proposed electron-transport sequence is consistent with the role of E3 as a 2e−/1e− switch and provides compelling evidence supporting a radical mechanism for E3-catalyzed C-3 deoxygenation. 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 biological deoxygenation.
AB - The CDP-6-deoxy-Δ3,4-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 purified enzyme is also a NADH oxidase capable of mediating the direct electron transfer from NADH to O2, forming H2O2. While previous work showed that E3 contains no common cofactor, one FAD and one plant ferredoxin type [2Fe-2S] center were found in this study to be associated with each molecule of E3. The iron-sulfur center is essential for E3 activity 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 reduction of FAD by NADH. The iron-sulfur cluster, receiving electrons one at a time from the reduced flavin, relays the reducing equivalents via another iron-sulfur center in the active site of E1 to its final acceptor, the E1-bound PMP-glucoseen adduct. The participation of a one-electron-carrying iron-sulfur center in this reduction is advantageous since both electrons are dispatched from the same redox state of the prosthetic group, allowing electrons of equal energy to be delivered to the final acceptor. This proposed electron-transport sequence is consistent with the role of E3 as a 2e−/1e− switch and provides compelling evidence supporting a radical mechanism for E3-catalyzed C-3 deoxygenation. 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 biological deoxygenation.
UR - https://www.scopus.com/pages/publications/0027515126
UR - https://www.scopus.com/pages/publications/0027515126#tab=citedBy
U2 - 10.1021/bi00095a025
DO - 10.1021/bi00095a025
M3 - Article
C2 - 8218267
AN - SCOPUS:0027515126
SN - 0006-2960
VL - 32
SP - 11934
EP - 11942
JO - Biochemistry
JF - Biochemistry
IS - 44
ER -