KINETIC CHARACTERIZATION OF AN ORGANIC RADICAL IN THE ASCARYLOSE BIOSYNTHETIC-PATHWAY

The lipopolysaccharide of Yersinia pseudotuberculosis V includes a 3,6 -dideoxyhexose, ascarylose, as the nonreducing end of the O-antigen te trasaccharide. The C-3 deoxygenation of CDP-6-deoxy-L-threo-D-glycero- 4-hexulose is a critical reaction in the biosynthesis of ascarylose. T he first half of the reaction is a dehydration catalyzed by CDP-6-deox y-L-threo-D-glycero-4-hexulose (E(1)), which is PMP-dependent and cont ains a redox-active [2Fe-2S] center. The second half is a reduction th at requires an additional enzyme, P-6-deoxy-L-threo-D-glycero-4-hexulo se-3-dehydrase reductase (E(3), formerly known as CDP-6-deoxy-Delta 3, 4-glucoseen reductase), which has a FAD and a [2Fe-2S] center in the a ctive site. Using NADH as the reductant in the coupled E(1)-E(3) react ion, we have monitored the kinetics of a radical intermediate using bo th stopped-flow spectrophotometry and rapid freeze-quench EPR under ae robic and hypoxic conditions. In the EPR studies, a sharp signal at g = 2.003 was found to appear at a rate which is kinetically competent, reaching its maximum intensity at similar to 150 ms. Stopped-flow UV-v is analysis of the reaction elucidated a minimum of six optically dist inguishable states in the mechanism of electron transfer from NADH to substrate. Interestingly, one of the detected intermediates has a time course nearly identical to that of the radical detected by rapid free ze-quench EPR. The difference UV-vis spectrum of this intermediate dis plays a maximum at 456 nm with a shoulder at 425 nm. Overall, these re sults are consistent with an electron transfer pathway that includes a radical intermediate with the unpaired spin localized on the substrat e-cofactor complex. Evidence in support of this mechanism is presented in this report. These studies add the PMP-glucoseen radical to the gr owing list of mechanistically important bioorganic radical intermediat es that have recently been discovered.