Journal
BIOCHEMISTRY
Volume 47, Issue 34, Pages 9007-9015Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi800419e
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Funding
- National Science Foundation [MCB-0238530]
- National Institutes of Health [R01-GM40313]
- NIH NRSA [F31-GM081979]
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The PduO-type ATP:corrinoid adenosyltransferase from Lactobacillus reitteri (LrPduO) catalyzes the formation of the essential Co-C bond of adenosylcobalamin (coenzyme B-12) 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 Collcobalamin (Co(1+)Cbi), our kinetic data obtained in this study provide in vitro evidence that LrPduO can in fact also utilize the incomplete corrinoid Co1+ cobinamide (Co(1+)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 Co(2+)Cbl and Co(2+)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 (similar to 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 outprevious 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 Co(2+)corrinoid intermediate that lacks any significant axial bonding interactions, to stabilize the redox-active, Co 3d(Z)(2)-based molecular orbital.
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