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

Abstract

IcmF is a 5'-deoxyadenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the carbon skeleton rearrangement of isobutyryl-CoA to butyryl-CoA. It is a bifunctional protein resulting from the fusion of a G-protein chaperone with GTPase activity and the cofactor- and substrate-binding mutase domains with isomerase activity. IcmF is prone to inactivation during catalytic turnover, thus setting up its dependence on a cofactor repair system. Herein, we demonstrate that the GTPase activity of IcmF powers the ejection of the inactive cob(II)alamin cofactor and requires the presence of an acceptor protein, adenosyltransferase, for receiving it. Adenosyltransferase in turn converts cob(II)alamin to AdoCbl in the presence of ATP and a reductant. The repaired cofactor is then reloaded onto IcmF in a GTPase-gated step. The mechanistic details of cofactor loading and offloading from the AdoCbl-dependent IcmF are distinct from those of the better characterized and homologous methylmalonyl-CoA mutase/G-protein chaperone system.

Keywords: ATP; GTPase; adenosylcobalamin (AdoCbl); low molecular weight G-protein; metalloprotein.

FIGURE 1.

Reaction mechanism and structure of IcmF. A, postulated mechanism for rearrangement of isobutyryl-CoA to butyryl-CoA. B, structure of C. metallidurans IcmF (PDB 4XC6) in which the B₁₂ (pink), G-domain (purple), and substrate (green) domains are highlighted in different colors. The linker region that contributes to the intersubunit interface is in orange, and B₁₂, GDP, and Mg²⁺ (cyan) ions are shown in sphere representation. C, close-up of the B₁₂-binding site in which His-39 ligation to the cobalt in AdoCbl can be seen.

FIGURE 2.

Binding of AdoCbl to ATR. A, changes in the UV-visible absorption spectrum upon binding of free AdoCbl (20 μm, black trace) to 3–30 μm ATR (final spectrum, red trace) in Buffer A. The absorption maximum shifts from 525 to 456 nm with isosbestic points at 388 and 481 nm. The data presented here are representative of at least three independent experiments. B, modulation of AdoCbl binding by nucleotides. The data represent the mean ± S.D. of three independent experiments.

FIGURE 3.

Binding of cob(II)alamin to ATR. A, changes in the UV-visible absorption spectrum of cob(II)alamin (20 μm) upon binding to ATR (60 μm) in Buffer B. Black trace, cob(II)alamin + ATR, λ_max = 470 nm; red trace, cob(II)alamin + ATR + 1 mm ATP, λ_max = 464 nm; blue trace, cob(II)alamin + ATR + 1 mm ATP + 600 μm Ti(III) citrate. The data are representative of at least three experiments. B, modulation of cob(II)alamin (10 μm) binding to ATR (1.8–90 μm) by nucleotides (1 mm). Unbound cob(II)alamin was separated by filtration and quantified by UV-visible spectroscopy. The data represent the mean ± S.D. of three independent experiments. C, CW-EPR spectrum of cob(II)alamin (300 μm) + ATR (450 μm) + 1 mm ATP (black trace) and simulated spectrum (red trace). Simulation parameters: g_x = 3.424, g_y = 2.479, and g_z = 1.792; A_x = 1147, A_y = 685, and A_z = 529 MHz; g-strain: σ_x = 0.177, σ_y = 0.0472, and σ_z = 0.0381.

FIGURE 4.

ATR-catalyzed conversion of cob(II)alamin to AdoCbl. A, HPLC analysis of samples from reactions containing cob(II)alamin (20 μm) and ATR (60 μm) in Buffer B. OH₂Cbl and AdoCbl elute at 18.3 and 25.7 min, respectively. Black trace, cob(II)alamin + ATR; red trace, cob(II)alamin + ATR + 1 mm ATP; blue trace, cob(II)alamin + ATR + 1 mm ATP + 600 μm Ti(III) citrate; dotted trace, cob(II)alamin + 1 mm ATP + 600 μm Ti(III) citrate. B, changes in the UV-visible absorption spectrum of cob(II)alamin (10 μm) mixed with ATR (15 μm) and ATP (0–90 μm). Inset, stoichiometry of cob(II)alamin (21 μm) binding to ATR (24 μm) in the presence of ATP (0–50 μm). The concentration of the ATR·cob(II)alamin·ATP complex was quantified by addition of 600 μm Ti(III) citrate followed by HPLC analysis of the AdoCbl formed. C, K_m for ATP was determined in reactions containing of ATR (0.3 μm), cob(II)alamin (50 μm), and 600 μm Ti(III) citrate. The data are representative of at least three experiments or the mean ± S.D. of three independent experiments.

FIGURE 5.

GTPase activity of IcmF is sensitive to the presence of cobalamin. The GTPase activity of IcmF (4 μm) in Buffer A at 25 °C was measured in the presence of 1 mm GTP ± cobalamins (20 μm). The data represent the mean ± S.D. of at least three independent experiments.

FIGURE 6.

Transfer of AdoCbl between ATR and IcmF. A, transfer of AdoCbl from ATR to IcmF. Upper, UV-visible spectra of AdoCbl (20 μm) bound to ATR (33 μm) or IcmF (20 μm); lower, changes in the UV-visible absorption spectrum of ATR (33 μm)·AdoCbl (20 μm) mixed with IcmF (20 μm) ± nucleotides (1 mm) in Buffer B. B, fraction of AdoCbl transferred from ATR to IcmF in the presence of 0–10 mm GDP, GTP, or GMPPCP, in the presence or absence of 1 mm ATP. C, fraction of AdoCbl remaining bound to IcmF when IcmF (20 μm)-bound AdoCbl (20 μm) was mixed with ATR (33 μm) in the presence of nucleotides. The data are representative of at least three experiments or the mean ± S.D. of three independent experiments.

FIGURE 7.

Ejection of cob(II)alamin from IcmF to ATR. A, changes in the UV-visible absorption spectrum of IcmF (20 μm) containing cob(II)alamin (20 μm) in Buffer B mixed with ATR (72 μm) and ATP (1 mm) in the presence of GTP (1 mm). Black traces, IcmF·cob(II)alamin; red traces, IcmF·cob(II)alamin + ATR·ATP; blue traces, IcmF·cob(II)alamin + ATR·ATP + Ti(III) citrate (600 μm). B, cob(II)alamin transfer from IcmF to ATR is enhanced by GTP. The fraction of cob(II)alamin transferred from IcmF to ATR was monitored in the presence of the indicated concentrations of nucleotide derivatives. C, minimal release of cob(II)alamin occurs from IcmF into solution. IcmF (20 μm) was mixed with cob(II)alamin (20 μm), and the fraction of cofactor released into solution in the presence of nucleotides (1 mm) was measured. D, dependence of cob(II)alamin (20 μm) transferred from IcmF (20 μm) to ATR (72 μm)·ATP (1 mm) on GTP concentration. The data presented here are representative of at least three experiments (D) or the mean ± S.D. of three independent experiments (B and C).

FIGURE 8.

Back-transfer of cob(II)alamin from ATR·ATP to IcmF. A, changes in the UV-visible absorption spectrum of ATR (33 μm)·cob(II)alamin (20 μm)·ATP (1 mm) mixed with IcmF (20 μm). Black trace, ATR·cob(II)alamin·ATP; red trace, ATR·cob(II)alamin·ATP + IcmF; blue trace, IcmF·cob(II)alamin. B, fraction of cob(II)alamin that remained bound to ATR following mixing of ATR·cob(II)alamin·ATP with IcmF in the presence of nucleotides (1 mm each). The data are representative of at least three experiments or the mean ± S.D. of three independent experiments.

FIGURE 9.

Proposed model for cofactor loading and repair of IcmF. Loading of the first equivalent of AdoCbl from ATR to IcmF occurs in the presence of ATP. Loading of the second equivalent AdoCbl from ATR to IcmF requires GTP hydrolysis of IcmF. Upon inactivation of AdoCbl, GTP hydrolysis is required for the transfer of the inactive cofactor, cob(II)alamin, from IcmF to ATR·ATP for repair.