Johnson D A, Gassner G T, Bandarian V, Ruzicka F J, Ballou D P, Reed G H, Liu H W
Department of Chemistry, University of Minnesota, Minneapolis 55455, USA.
Biochemistry. 1996 Dec 10;35(49):15846-56. doi: 10.1021/bi961370w.
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.
假结核耶尔森氏菌V的脂多糖包含一种3,6-二脱氧己糖——蛔糖，作为O抗原四糖的非还原端。CDP-6-脱氧-L-苏式-D-甘油-4-己酮糖的C-3脱氧反应是蛔糖生物合成中的关键反应。该反应的前半部分是由CDP-6-脱氧-L-苏式-D-甘油-4-己酮糖-3-脱水酶（E1）催化的脱水反应，E1依赖于磷酸吡哆醛且含有一个氧化还原活性的[2Fe-2S]中心。后半部分是还原反应，需要另一种酶——CDP-6-脱氧-L-苏式-D-甘油-4-己酮糖-3-脱水酶还原酶（E3，以前称为CDP-6-脱氧-δ3,4-葡萄糖烯还原酶），其活性位点含有一个黄素腺嘌呤二核苷酸（FAD）和一个[2Fe-2S]中心。在偶联的E1-E3反应中使用NADH作为还原剂，我们在有氧和低氧条件下，通过停流分光光度法和快速冷冻淬灭电子顺磁共振（EPR）监测了一种自由基中间体的动力学。在EPR研究中，发现g = 2.003处的一个尖锐信号以动力学上可行的速率出现，并在大约150毫秒时达到其最大强度。对该反应的停流紫外可见分析揭示了从NADH到底物的电子转移机制中至少有六个光学上可区分的状态。有趣的是，检测到的一种中间体的时间进程与通过快速冷冻淬灭EPR检测到的自由基的时间进程几乎相同。该中间体的差示紫外可见光谱在456 nm处有一个最大值，在425 nm处有一个肩峰。总体而言，这些结果与一个电子转移途径一致，该途径包括一个自由基中间体，其未成对自旋定域在底物-辅因子复合物上。本报告提供了支持该机制的证据。这些研究将磷酸吡哆醛-葡萄糖烯自由基添加到最近发现的越来越多的具有重要机制意义的生物有机自由基中间体名单中。
