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

Abstract

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 co-substrate 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 pre-incubated 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, so as to stabilize the redox-active, Co 3dz2-based molecular orbital.

Diverse Bacterial Microcompartment Organelles

“…In vitro studies indicate that the substrate of PduS is cob(II)alamin bound to an ATR. When cob(II)alamin binds ATR, it undergoes a transition to the 4-coordinate base-off conformer (132)(133)(134)(135). Transition to this state raises the midpoint potential of the cob(II)alamin/cob(I)alamin couple by about 250 mV, facilitating reduction.…”

Dihydroflavin-driven Adenosylation of 4-Coordinate Co(II) Corrinoids

“…1). In this process, ACA enzymes bind 5-coordinate cob(II)alamin and displace the lower ligand 5,6-dimethylbenzimidazole, resulting in a 4-coordinate cob(II)alamin intermediate that lacks axial ligands (17)(18)(19)(20)(21)(22). In this 4-coordinate cob(II)alamin intermediate, the 3d z 2 orbital of the cobalt ion is stabilized, raising the reduction potential ϳ250 mV and bringing the reduction potential of Co 2ϩ/ϩ Cbl to within physiological range (20).…”

The EutT Enzyme of Salmonella enterica Is a Unique ATP:Cob(I)alamin Adenosyltransferase Metalloprotein That Requires Ferrous Ions for Maximal Activity

“…oenzyme B 12 (adenosylcobalamin, AdoCbl, CoB 12 ) is an essential nutrient for animals, lower eukaryotes, and many prokaryotes. The unique organometallic bond of AdoCbl, between the cobalt ion of the corrinoid and the carbon of the 5=-deoxyadenosyl group, lies at the center of its reactivity.…”

“…These two types of enzymes facilitate the thermodynamically unfavorable reduction of cob(II)alamin to cob(I)alamin by generating a cob(II)alamin four-coordinate intermediate in the active site of the enzyme (10)(11)(12). Both CobA and PduO use conserved aromatic side chains located directly below the cobalt ion to displace the lower ligand of Cbl and generate the four-coordinate species (5,13).…”