title: 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-delta 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 approximately 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.
authors.affiliation: Department of Chemistry, University of Minnesota, Minneapolis 55455, USA.
authors.forename: D A
authors.initials: DA
authors.lastname: Johnson
authors.forename: G T
authors.initials: GT
authors.lastname: Gassner
authors.forename: V
authors.initials: V
authors.lastname: Bandarian
authors.forename: F J
authors.initials: FJ
authors.lastname: Ruzicka
authors.forename: D P
authors.initials: DP
authors.lastname: Ballou
authors.forename: G H
authors.initials: GH
authors.lastname: Reed
authors.forename: H W
authors.initials: HW
authors.lastname: Liu
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D003565:Cytidine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000378:metabolism; D006601:Hexoses / Q000096:biosynthesis*; D007700:Kinetics; D008070:Lipopolysaccharides / Q000737:chemistry; D008956:Models, Chemical; D015394:Molecular Structure; D009243:NAD / Q000378:metabolism; D019081:O Antigens / Q000737:chemistry; D010088:Oxidoreductases / Q000378:metabolism; D013053:Spectrophotometry; D015011:Yersinia pseudotuberculosis / Q000201:enzymology* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D005609:Free Radicals; D006601:Hexoses; D008070:Lipopolysaccharides; D019081:O Antigens; C090489:cytidine diphosphate-6-deoxyxylo-4-hexulose; D009243:NAD; C009616:tyvelose; D003565:Cytidine Diphosphate; D010088:Oxidoreductases