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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• 作者：Kiyoung Park ; Paola E. Mera ; Jorge C. Escalante-Semerena ; Thomas C. Brunold
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：August 26, 2008
• 年：2008
• 卷：47
• 期：34
• 页码：9007-9015
• 全文大小：309K
• 年卷期：v.47,no.34(August 26, 2008)
• ISSN：1520-4995

文摘

The PduO-type ATP:corrinoid adenosyltransferase from Lactobacillus 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 that LrPduO can in fact also utilize the incomplete corrinoid Co¹⁺cobinamide (Co¹⁺Cbi) as an alternative substrate. To explore the mechanism by which LrPduO 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 to LrPduO 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 3d_z²-based molecular orbital.