Kinetic Characterization of an Organic Radical in the Ascarylose Biosynthetic Pathway

Abstract

The lipopolysaccharide of Yersinia pseudotuberculosis V includes a 3,6-dideoxyhexose, ascarylose, as the nonreducing end of the O-antigen tetrasaccharide. The C-3 deoxygenation of CDP-6-deoxy-l-threo-d-glycero-4-hexulose is a critical reaction in the biosynthesis of ascarylose. The first half of the reaction is a dehydration catalyzed by CDP-6-deoxy-l-threo-d-glycero-4-hexulose-3-dehydrase (E₁), which is PMP-dependent and contains a redox-active [2Fe-2S] center. The second half is a reduction that requires an additional enzyme, CDP-6-deoxy-l-threo-d-glycero-4-hexulose-3-dehydrase reductase (E₃, formerly known as CDP-6-deoxy-Δ^3,4-glucoseen reductase), which has a FAD and a [2Fe-2S] center in the active site. Using NADH as the reductant in the coupled E₁−E₃ reaction, we have monitored the kinetics of a radical intermediate using both stopped-flow spectrophotometry and rapid freeze−quench EPR under aerobic 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 ∼150 ms. Stopped-flow UV−vis analysis of the reaction elucidated a minimum of six optically distinguishable 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 freeze−quench EPR. The difference UV−vis spectrum of this intermediate displays a maximum at 456 nm with a shoulder at 425 nm. Overall, these results are consistent with an electron transfer pathway that includes a radical intermediate with the unpaired spin localized on the substrate−cofactor complex. Evidence in support of this mechanism is presented in this report. These studies add the PMP−glucoseen radical to the growing list of mechanistically important bioorganic radical intermediates that have recently been discovered.