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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.

G-protein metallochaperone MeaB in bacteria [methylmalonic aciduria type A (MMAA) in humans] is responsible for facilitating the delivery of adenosylcobalamin (AdoCbl) to methylmalonyl-CoA mutase (MCM), the only AdoCbl-dependent enzyme in humans. Genetic defects in the switch III region of MMAA lead to the genetic disorder methylmalonic aciduria in which the body is unable to process certain lipids. Here, we present a crystal structure of Methylobacterium extorquens MeaB bound to a nonhydrolyzable guanosine triphosphate (GTP) analog guanosine-5′-[(β,γ)-methyleno]triphosphate (GMPPCP) with the Cbl-binding domain of its target mutase enzyme (MeMCMcbl). This structure provides an explanation for the stimulation of the GTP hydrolyase activity of MeaB afforded by target protein binding. We find that upon MCMcbl association, one protomer of the MeaB dimer rotates ~180°, such that the inactive state of MeaB is converted to an active state in which the nucleotide substrate is now surrounded by catalytic residues. Importantly, it is the switch III region that undergoes the largest change, rearranging to make direct contacts with the terminal phosphate of GMPPCP. These structural data additionally provide insights into the molecular basis by which this metallochaperone contributes to AdoCbl delivery without directly binding the cofactor. Our data suggest a model in which GTP-bound MeaB stabilizes a conformation of MCM that is open for AdoCbl insertion, and GTP hydrolysis, as signaled by switch III residues, allows MCM to close and trap its cofactor. Substitutions of switch III residues destabilize the active state of MeaB through loss of protein:nucleotide and protein:protein interactions at the dimer interface, thus uncoupling GTP hydrolysis from AdoCbl delivery.

Control over transition metal redox state is essential for metalloprotein function and can be achieved via coordination chemistry and/or sequestration from bulk solvent. Human methylmalonyl-Coenzyme A (CoA) mutase (MCM) catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA using 5′-deoxyadenosylcobalamin (AdoCbl) as a metallocofactor. During catalysis, the occasional escape of the 5′-deoxyadenosine (dAdo) moiety leaves the cob(II)alamin intermediate stranded and prone to hyperoxidation to hydroxocobalamin, which is recalcitrant to repair. In this study, we have identified the use of bivalent molecular mimicry by ADP, coopting the 5′-deoxyadenosine and diphosphate moieties in the cofactor and substrate, respectively, to protect against cob(II)alamin overoxidation on MCM. Crystallographic and electron paramagnetic resonance (EPR) data reveal that ADP exerts control over the metal oxidation state by inducing a conformational change that seals off solvent access, rather than by switching five-coordinate cob(II)alamin to the more air stable four-coordinate state. Subsequent binding of methylmalonyl-CoA (or CoA) promotes cob(II)alamin off-loading from MCM to adenosyltransferase for repair. This study identifies an unconventional strategy for controlling metal redox state by an abundant metabolite to plug active site access, which is key to preserving and recycling a rare, but essential, metal cofactor.

Methylmalonic acidemia (MMA) is a genetic condition associated with intellectual disability and a high mortality rate. It is caused by pathogenic variants in the MMUT gene, which codes methylmalonyl-CoA mutase enzyme (MUT). In the Mexican population, the variant NM_000255.4:c.322C>T or p.(Arg108Cys) is the most frequently found, but its structural pathogenic effect is scarcely studied. To describe the clinical picture of p.(Arg108Cys) homozygous patients and to predict its structural pathogenic effect, we performed an analysis of the medical files from six MMA Mexican p.(Arg108Cys) homozygous patients. The structural changes in MUT caused by this variant were analyzed through molecular dynamics simulations (MDS) and docking and compared with the wild-type (Wt) enzyme. The main clinical symptoms presented by the patients were feeding difficulties, lethargy, and neurodevelopmental delay, with a predominance of early-onset phenotype and a mortality rate of 83%. We found significant structural changes in MUT structure, particularly in the catalytic domain, with increased volume cavity, shortening of the binding substrate tunnel, and aberrant accommodation. Also, the dimerization interface area increased from 1343 Å2 in the Wt to 3386 Å2, and the dimer formation involved a different set of amino acids. The NM_000255.4:c.322C>T or p.(Arg108Cys) MMUT variant is associated with a severe outcome in MMA Mexican patients, and the enzyme was associated with ostentatious topological changes in the secondary and tertiary structure, which impacted the catalytic domain, the accommodation of the substrate, and the dimerization interface. Further ex vivo functional studies are needed to confirm these predictions, such as enzymatic activity measurements in fibroblasts of patients.

Vitamin B 12 is an organometallic compound with important metabolic derivatives that act as cofactors of certain enzymes,which have been grouped into three subfamilies depending on their cofactors.Among them,methylmalonyl-CoA mutase （MCM） has been extensively studied.This enzyme catalyzes the reversible isomerization of L-methylmalonyl-CoA to succinyl-CoA using adenosylcobalamin （AdoCbl） as a cofactor participating in the generation of radicals that allow isomerization of the substrate.The crystal structure of MCM determined in Propionibacterium freudenreichii var.shermanii has helped to elucidate the role of this cofactor AdoCbl in the reaction to specify the mechanism by which radicals are generated from the coenzyme and to clarify the interactions between the enzyme,coenzyme,and substrate.The existence of human methylmalonic acidemia （MMA） due to the presence of mutations in MCM shows the importance of its role in metabolism.The recent crystallization of the human MCM has shown that despite being similar to the bacterial protein,there are significant differences in the structural organization of the two proteins.Recent studies have identified the involvement of an accessory protein called MMAA,which interacts with MCM to prevent MCM＇s inactivation or acts as a chaperone to promote regeneration of inactivated enzyme.The interdisciplinary studies using this protein as a model in different organisms have helped to elucidate the mechanism of action of this isomerase,the impact of mutations at a functional level and their repercussion in the development and progression of MMA in humans.It is still necessary to study the mechanisms involved in more detail using new methods.

Significance Metalloproteins are ubiquitous, accounting for about 30–50% of all proteins. Their functions are wide-ranging, but metalloproteins are frequently used to carry out challenging molecular transformations. Metalloprotein reactivity comes at a price, however, often requiring specialized molecular machinery for holoenzyme assembly. G-protein metallochaperones are an important part of this assembly apparatus, but an understanding of their molecular mechanisms has been hindered by a lack of structural data. Here, we describe crystal structures of a G-protein metallochaperone together with a target enzyme, in this case an adenosylcobalamin-dependent radical enzyme, thereby providing a visualization of the molecular architecture of the G-protein:target enzyme complex. G-protein metallochaperones ensure fidelity during cofactor assembly for a variety of metalloproteins, including adenosylcobalamin (AdoCbl)-dependent methylmalonyl-CoA mutase and hydrogenase, and thus have both medical and biofuel development applications. Here, we present crystal structures of IcmF, a natural fusion protein of AdoCbl-dependent isobutyryl-CoA mutase and its corresponding G-protein chaperone, which reveal the molecular architecture of a G-protein metallochaperone in complex with its target protein. These structures show that conserved G-protein elements become ordered upon target protein association, creating the molecular pathways that both sense and report on the cofactor loading state. Structures determined of both apo- and holo-forms of IcmF depict both open and closed enzyme states, in which the cofactor-binding domain is alternatively positioned for cofactor loading and for catalysis. Notably, the G protein moves as a unit with the cofactor-binding domain, providing a visualization of how a chaperone assists in the sequestering of a precious cofactor inside an enzyme active site.

Significance The origin of the catalytic power of B ₁₂ enzymes has been a major puzzle despite our previous finding that this effect is due to electrostatic stabilization of the leaving group. Recent findings of very large entropic contributions to catalysis were presented as an alternative to the electrostatic idea. Here, we use our ability to evaluate entropic contributions by the restraint release (RR) approach to reexamine the nature of the catalytic effect. The RR approach reproduces the observed entropic contributions to the activation barrier and demonstrates that the entropic effect is due to the previously identified electrostatic factors. Thus, we have further substantiated our paradigm for the origin of the catalytic power of B ₁₂ enzymes. The catalytic power of enzymes containing coenzyme B ₁₂ has been, in some respects, the “last bastion” for the strain hypothesis. Our previous study of this system established by a careful sampling that the major part of the catalytic effect is due to the electrostatic interaction between the ribose of the ado group and the protein and that the strain contribution is very small. This finding has not been sufficiently appreciated due to misunderstandings of the power of the empirical valence bond (EVB) calculations and the need of sufficient sampling. Furthermore, some interesting new experiments point toward entropic effects as the source of the catalytic power, casting doubt on the validity of the electrostatic idea, at least, in the case of B ₁₂ enzymes. Here, we focus on the observation of the entropic effects and on analyzing their origin. We clarify that our EVB approach evaluates free energies rather than enthalpies and demonstrate by using the restraint release (RR) approach that the observed entropic contribution to the activation barrier is of electrostatic origin. Our study illustrates the power of the RR approach by evaluating the entropic contributions to catalysis and provides further support to our paradigm for the origin of the catalytic power of B ₁₂ enzymes. Overall, our study provides major support to our electrostatic preorganization idea and also highlights the basic requirements from ab initio quantum mechanics/molecular mechanics calculations of activation free energies of enzymatic reactions.

Heterotrimeric G proteins contain a switch III motif that regulates enzyme function. Structural and biochemical studies now identify a similar switch III loop in a nonheterotrimeric G-protein chaperone that explains the debilitating effects of mutations linked to methylmalonic aciduria. Fidelity during cofactor assembly is essential for the proper functioning of metalloenzymes and is ensured by specific chaperones. MeaB, a G-protein chaperone for the coenzyme B 12 -dependent radical enzyme methylmalonyl-CoA mutase (MCM), uses the energy of GTP binding, hydrolysis or both to regulate cofactor loading into MCM, protect MCM from inactivation and rescue MCM that is inactivated during turnover. Typically, G proteins signal to client proteins using the conformationally mobile switch I and II loops. Crystallographic snapshots of MeaB reported herein reveal a new switch III element that has substantial conformational plasticity. Using alanine-scanning mutagenesis, we demonstrate that the switch III motif is critical for bidirectional signal transmission of the GTPase-activating protein activity of MCM and the chaperone functions of MeaB in the MeaB–MCM complex. Mutations in the switch III loop identified in patients corrupt this interprotein communication and lead to methylmalonic aciduria, an inborn error of metabolism.

Significant accumulation of the methylmalonyl-CoA mutase apoenzyme was observed in the photosynthetic flagellate Euglena gracilis Z at the end of the logarithmic growth phase. The apoenzyme was converted to a holoenzyme by incubation for 4 h at 4°C with 10 μM 5′-deoxyadenosylcobalamin, and then, the holoenzyme was purified to homogeneity and characterized. The apparent molecular mass of the enzyme was calculated to be 149.0 kDa ± 5.0 kDa using Superdex 200 gel filtration. SDS-polyacrylamide gel electrophoresis of the purified enzyme yielded a single protein band with an apparent molecular mass of 75.0 kDa ± 3.0 kDa, indicating that the Euglena enzyme is composed of two identical subunits. The purified enzyme contained one mole of prosthetic 5′-deoxyadenosylcobalamin per mole of the enzyme subunit. Moreover, we cloned the full-length cDNA of the Euglena enzyme. The cDNA clone contained an open reading frame encoding a protein of 717 amino acids with a calculated molecular mass of 78.3 kDa, preceded by a putative mitochondrial targeting signal consisting of nine amino acid residues. Furthermore, we studied some properties and physiological function of the Euglena enzyme.

B12-dependent methylmalonyl-CoA mutase catalyses the interchange of a hydrogen atom and the carbonyl-CoA group on adjacent carbons of methylmalonyl-CoA to give the rearranged product, succinyl-CoA. The first step in this reaction involves the transient generation of cofactor radicals by homolytic rupture of the cobalt-carbon bond to generate the deoxyadenosyl radical and cob(II)alamin. This step exhibits a curious sensitivity to isotopic substitution in the substrate, methylmalonyl-CoA, which has been interpreted as evidence for kinetic coupling. The magnitude of the isotopic discrimination is large and a deuterium isotope effect ranging from 35.6 at 20 °C to 49.9 at 5 °C has been recorded. Arrhenius analysis of the temperature dependence of this isotope effect provides evidence for quantum tunnelling in this hydrogen transfer step. The mechanistic complexity of the observed rate constant for cobalt-carbon bond homolysis together with the spectroscopically silent nature of many of the component steps limits the insights that can be derived by experimental approaches alone. Computational studies using a newly developed geometry optimization scheme that allows determination of the transition state in the full quantum mechanical/molecular mechanical coordinate space have yielded novel insights into the strategy deployed for labilizing the cobalt-carbon bond and poising the resulting deoxyadenosyl radical for subsequent hydrogen atom abstraction.