10.1021\/bi961370w<\/a><\/p>\nAuthors:<\/h4>\n
David A. Johnson, George Gassner, Vahe Bandarian, Frank J. Ruzicka, David P. Ballou, George H. Reed, Hung\u2010wen Liu<\/p>\n
Abstract:<\/h4>\n
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.<\/p>\n","protected":false},"excerpt":{"rendered":"
DOI: 10.1021\/bi961370w Authors: David A. Johnson, George Gassner, Vahe Bandarian, Frank J. Ruzicka, David P. Ballou, George H. Reed, Hung\u2010wen Liu 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 […]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[5],"tags":[],"class_list":["post-677","post","type-post","status-publish","format-standard","hentry","category-freeze-quench"],"_links":{"self":[{"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=\/wp\/v2\/posts\/677","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=677"}],"version-history":[{"count":0,"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=\/wp\/v2\/posts\/677\/revisions"}],"wp:attachment":[{"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=677"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=677"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/citations.tools.bio-logic.fr\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=677"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}