Structural Characterization of a Human-Type Corrinoid Adenosyltransferase Confirms That Coenzyme B₁₂ Is Synthesized through a Four-Coordinate Intermediate

Abstract

ATP:cob(I)alamin adenosyltransferases (ACAs) catalyze the transfer of the 5'-deoxyadenosyl moiety from ATP to the upper axial ligand position of cobalamin in the synthesis of coenzyme B 12. For the ACA-catalyzed reaction to proceed, cob(II)alamin must be reduced to cob(I)alamin in the enzyme active site. This reduction is facilitated through the generation of a four-coordinate cob(II)alamin intermediate on the enzyme. We have determined the high-resolution crystal structure of a human-type ACA from Lactobacillus reuteri with a four-coordinate cob(II)alamin bound in the enzyme active site and with the product, adenosylcobalamin, partially occupied in the active site. The assembled structures represent snapshots of the steps in the ACA-catalyzed formation of the cobalt-carbon bond of coenzyme B 12. The structures define the corrinoid binding site and provide visual evidence for a base-off, four-coordinate cob(II)alamin intermediate. The complete structural description of ACA-mediated catalysis reveals the molecular features of four-coordinate cob(II)alamin stabilization and provides additional insights into the molecular basis for dysfunction in human patients suffering from methylmalonic aciduria.

Cofactor Editing by the G-protein Metallochaperone Domain Regulates the Radical B12 Enzyme IcmF

“…The adenosine groups of ATP and of AdoCbl occupy the same position in the ATR site (29,30), and their binding is mutually exclusive. As expected, in the presence of ATP, a higher ratio of ATR/AdoCbl is needed to saturate the cobalamin-binding site (Fig.…”

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).…”

“…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). Recent structural and kinetic analyses revealed that a conserved phenylalanine in the active site of the PduOtype ACA enzyme is critical to the formation of the 4-coordinate cob(II)alamin intermediate (17,21).…”

“…Formation (17,19,21,22). The conserved residue Phe-112, located in the active site of PduO, is critical for the displacement of the lower ligand base of cob(II)alamin (17, 21).…”

Characterization of the PduS Cobalamin Reductase of Salmonella enterica and Its Role in the Pdu Microcompartment

“…Next, cob(II)alamin is reduced to cob(I)alamin, and ATP:cob(I)alamin adenosyltransferase (ATR) transfers a 5Ј deoxyadenosyl group from ATP to cob(I)alamin to form AdoCbl (10,11,28,29,35,63,64,72). Studies indicate that prior to reduction cob(II)alamin binds the ATR and undergoes a transition to the 4-coordinate base-off conformer (41,48,59,61,62). Transition to the 4-coordinate state raises the midpoint potential of the cob(II)alamin/cob(I)alamin couple by about 250 mV, facilitating reduction (60).…”