Allosteric Regulation of Oligomerization by a B12 Trafficking G-Protein Is Corrupted in Methylmalonic Aciduria

Cobalamin is a complex organometallic cofactor that is processed and targeted via a network of chaperones to its dependent enzymes. AdoCbl (5′-deoxyadenosylcobalamin) is synthesized from cob(II)alamin in a reductive adenosylation reaction catalyzed by adenosyltransferase (ATR), which also serves as an escort, delivering AdoCbl to methylmalonyl-CoA mutase (MCM). The mechanism by which ATR signals that its cofactor cargo is ready (AdoCbl) or not [cob(II)alamin] for transfer to MCM, is not known. In this study, we have obtained crystallographic snapshots that reveal ligand-induced ordering of the N terminus ofMycobacterium tuberculosisATR, which organizes a dynamic cobalamin binding site and exerts exquisite control over coordination geometry, reactivity, and solvent accessibility. Cob(II)alamin binds with its dimethylbenzimidazole tail splayed into a side pocket and its corrin ring buried. The cosubstrate, ATP, enforces a four-coordinate cob(II)alamin geometry, facilitating the unfavorable reduction to cob(I)alamin. The binding mode for AdoCbl is notably different from that of cob(II)alamin, with the dimethylbenzimidazole tail tucked under the corrin ring, displacing the N terminus of ATR, which is disordered. In this solvent-exposed conformation, AdoCbl undergoes facile transfer to MCM. The importance of the tail in cofactor handover from ATR to MCM is revealed by the failure of 5′-deoxyadenosylcobinamide, lacking the tail, to transfer. In the absence of MCM, ATR induces a sacrificial cobalt–carbon bond homolysis reaction in an unusual reversal of the heterolytic chemistry that was deployed to make the same bond. The data support an important role for the dimethylbenzimidazole tail in moving the cobalamin cofactor between active sites.

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“…6). The transfer of AdoCbl to MCM is gated by the GTPase activity of yet another chaperone, CblA (18,36).…”

Section: Discussionmentioning

confidence: 99%

Mobile loop dynamics in adenosyltransferase control binding and reactivity of coenzyme B ₁₂

Mascarenhas

Ruetz

McDevitt

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…To gain insights into why the repair process is impeded by I-CoA but not M-CoA, we used hMCM, which forms a stable complex with CblA when it is in need of repair but is free in the active AdoCbl-bound state (fig. S12A) (27). In size exclusion chromatography profiles, hMCM is a stand-alone dimer (173 kDa) in the presence of M-CoA and the repair mixture (fig.…”

Section: I-coa Inhibits MCM Repairmentioning

confidence: 99%

Itaconyl-CoA forms a stable biradical in methylmalonyl-CoA mutase and derails its activity and repair

Ruetz

Campanello

Purchal

et al. 2019

Science

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Itaconate is an immunometabolite with both anti-inflammatory and bactericidal effects. Its coenzyme A (CoA) derivative, itaconyl-CoA, inhibits B12-dependent methylmalonyl-CoA mutase (MCM) by an unknown mechanism. We demonstrate that itaconyl-CoA is a suicide inactivator of human and Mycobacterium tuberculosis MCM, which forms a markedly air-stable biradical adduct with the 5′-deoxyadenosyl moiety of the B12 coenzyme. Termination of the catalytic cycle in this way impairs communication between MCM and its auxiliary repair proteins. Crystallography and spectroscopy of the inhibited enzyme are consistent with a metal-centered cobalt radical ~6 angstroms away from the tertiary carbon-centered radical and suggest a means of controlling radical trajectories during MCM catalysis. Mycobacterial MCM thus joins enzymes in the glyoxylate shunt and the methylcitrate cycle as targets of itaconate in pathogen propionate metabolism.

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“…The mitochondrial pathway leads to the synthesis of 5′-deoxyadenosylcobalamin (AdoCbl) and includes chaperones encoded by the cbl A and cbl B loci. The cbl A and cbl B loci encode a GTPase ( 7 ) and adenosyltransferase (ATR) ( 8 ), respectively, which play distinct roles in cofactor assimilation, loading, and repair of MCM ( 9 , 10 , 11 , 12 ).…”

mentioning

confidence: 99%

“…1 ). It can be transferred directly to MCM ( 9 , 10 , 19 , 20 ) where it is bound in a six-coordinate base-off/His on conformation ( 21 ). When MCM is not available to accept AdoCbl, ATR catalyzes the homolysis of the newly formed Co-carbon bond in the presence of PPPi, the co-product ( 10 ).…”

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confidence: 99%

“…Occasional inactivation resulting from loss of the deoxyadenosyl intermediate leads to the failure to reform AdoCbl at the end of the catalytic cycle and to accumulation of inactive cob(II)alamin in MCM, which needs repair ( 23 ). Cob(II)alamin is translocated from MCM to ATR in a cofactor off-loading process that is driven by GTP hydrolysis catalyzed by the CblA chaperone ( 9 ).…”

mentioning

confidence: 99%

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Patient mutations in human ATP:cob(I)alamin adenosyltransferase differentially affect its catalytic versus chaperone functions

Gouda

Mascarenhas

Pillay

et al. 2021

Journal of Biological Chemistry

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Human ATP:cob(I)alamin adenosyltransferase (ATR) is a mitochondrial enzyme that catalyzes an adenosyl transfer to cob(I)alamin, synthesizing 5′-deoxyadenosylcobalamin (AdoCbl) or coenzyme B 12 . ATR is also a chaperone that escorts AdoCbl, transferring it to methylmalonyl-CoA mutase, which is important in propionate metabolism. Mutations in ATR lead to methylmalonic aciduria type B, an inborn error of B 12 metabolism. Our previous studies have furnished insights into how ATR protein dynamics influence redox-linked cobalt coordination chemistry, controlling its catalytic versus chaperone functions. In this study, we have characterized three patient mutations at two conserved active site residues in human ATR, R190C/H, and E193K and obtained crystal structures of R190C and E193K variants, which display only subtle structural changes. All three mutations were found to weaken affinities for the cob(II)alamin substrate and the AdoCbl product and increase K M(ATP) . 31 P NMR studies show that binding of the triphosphate product, formed during the adenosylation reaction, is also weakened. However, although the k cat of this reaction is significantly diminished for the R190C/H mutants, it is comparable with the WT enzyme for the E193K variant, revealing the catalytic importance of Arg-190. Furthermore, although the E193K mutation selectively impairs the chaperone function by promoting product release into solution, its catalytic function might be unaffected at physiological ATP concentrations. In contrast, the R190C/H mutations affect both the catalytic and chaperoning activities of ATR. Because the E193K mutation spares the catalytic activity of ATR, our data suggest that the patients carrying this mutation are more likely to be responsive to cobalamin therapy.

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Allosteric Regulation of Oligomerization by a B12 Trafficking G-Protein Is Corrupted in Methylmalonic Aciduria

Cited by 16 publications

References 49 publications

Mobile loop dynamics in adenosyltransferase control binding and reactivity of coenzyme B ₁₂

Mobile loop dynamics in adenosyltransferase control binding and reactivity of coenzyme B ₁₂

Itaconyl-CoA forms a stable biradical in methylmalonyl-CoA mutase and derails its activity and repair

Patient mutations in human ATP:cob(I)alamin adenosyltransferase differentially affect its catalytic versus chaperone functions

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Allosteric Regulation of Oligomerization by a B12 Trafficking G-Protein Is Corrupted in Methylmalonic Aciduria

Cited by 16 publications

References 49 publications

Mobile loop dynamics in adenosyltransferase control binding and reactivity of coenzyme B 12

Mobile loop dynamics in adenosyltransferase control binding and reactivity of coenzyme B 12

Itaconyl-CoA forms a stable biradical in methylmalonyl-CoA mutase and derails its activity and repair

Patient mutations in human ATP:cob(I)alamin adenosyltransferase differentially affect its catalytic versus chaperone functions

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Product

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Mobile loop dynamics in adenosyltransferase control binding and reactivity of coenzyme B ₁₂

Mobile loop dynamics in adenosyltransferase control binding and reactivity of coenzyme B ₁₂