Kinetic and spectroscopic studies of the ATP:corrinoid adenosyltransferase PduO from Lactobacillus reuteri: substrate specificity and insights into the mechanism of Co(II)corrinoid reduction.
Kinetic and spectroscopic studies of the ATP:corrinoid adenosyltransferase PduO from Lactobacillus reuteri: substrate specificity and insights into the mechanism of Co(II)corrinoid reduction.
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罗伊氏乳杆菌 ATP：类咕啉腺苷转移酶 PduO 的动力学和光谱研究：底物特异性和 Co(II) 类咕啉还原机制的见解。
DOI：
10.1021/bi800419e
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发表时间：
2008
期刊：
影响因子：
2.9
通讯作者：
Brunold,ThomasC
中科院分区：
文献类型：
--
作者：
Park,Kiyoung;Mera,PaolaE;Escalante-Semerena,JorgeC;Brunold,ThomasC
来自罗伊氏乳杆菌的PduO型ATP：类可啉腺苷转移酶（LrPduO）通过将腺苷基团从辅助底物ATP转移到酶活性位点产生的瞬时Co 1+类可啉物质，催化腺苷钴胺素（辅酶B12）的必需Co-C键的形成。虽然PduO型酶以前被认为只能腺苷化Co 1+钴胺素（Co 1 +Cbl−），但我们在本研究中获得的动力学数据提供了体外证据，证明LrPduO实际上也可以利用不完全的类咕啉Co 1 +cobinamide（Co 1 +Cbi）作为替代底物。为了探索LrPduO克服其Co2+corrinoid底物的还原的机制，我们研究了酶活性位点如何通过使用电子吸收，磁性圆二色性和电子顺磁共振光谱技术改变Co2+Cbl和Co2+Cbi+的几何和电子性质。我们的数据显示，在与ATP预孵育的LrPduO结合后，两种Co2+类可啉都经历了部分（约40 - 50%）转化为不同的顺磁性Co2+物质。这些物种的光谱特征是一致的，基本上是四坐标，正方形平面的Co 2+配合物，根据我们以前的研究中获得的结果进行比较相关的酶。因此，腺苷转移酶进行Co 2 +→ Co 1+还原的一般策略似乎涉及形成一种“活化的”Co 2+类可啉中间体，该中间体缺乏任何显著的轴向键合相互作用，以稳定氧化还原活性的Co 3dz 2-基分子轨道。
The PduO-type ATP:corrinoid adenosyltransferase fromLactobacillus reuteri(LrPduO) catalyzes the formation of the essential Co−C bond of adenosylcobalamin (coenzyme B12) by transferring the adenosyl group from cosubstrate ATP to a transient Co1+corrinoid species generated in the enzyme active site. While PduO-type enzymes have previously been believed to be capable of adenosylating only Co1+cobalamin (Co1+Cbl−), our kinetic data obtained in this study provide in vitro evidence thatLrPduO can in fact also utilize the incomplete corrinoid Co1+cobinamide (Co1+Cbi) as an alternative substrate. To explore the mechanism by whichLrPduO overcomes the thermodynamically challenging reduction of its Co2+corrinoid substrates, we have examined how the enzyme active site alters the geometric and electronic properties of Co2+Cbl and Co2+Cbi+by using electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopic techniques. Our data reveal that upon binding toLrPduO that was preincubated with ATP, both Co2+corrinoids undergo a partial (∼40−50%) conversion to distinct paramagnetic Co2+species. The spectroscopic signatures of these species are consistent with essentially four-coordinate, square-planar Co2+complexes, based on a comparison with the results obtained in our previous studies of related enzymes. Consequently, it appears that the general strategy employed by adenosyltransferases for effecting Co2+→ Co1+reduction involves the formation of an “activated” Co2+corrinoid intermediate that lacks any significant axial bonding interactions, to stabilize the redox-active, Co 3dz2-based molecular orbital.