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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 of Mycobacterium tuberculosis ATR, 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.

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.

Commensal and pathogenic bacteria must deal with many different stress conditions to survive in and colonize the human gastrointestinal tract. One major challenge that bacteria encounter in the gut is the high concentration of bile salts, which not only aid in food absorption but also act as effective physiological antimicrobials. The mechanism by which bile salts limit bacterial growth is still largely unknown. Here, we show that bile salts cause widespread protein unfolding and aggregation, affecting many essential proteins. Simultaneously, the bacterial cytosol becomes highly oxidizing, indicative of disulfide stress. Strains defective in reducing oxidative thiol modifications, restoring redox homeostasis, or preventing irreversible protein aggregation under disulfide stress conditions are sensitive to bile salt treatment. Surprisingly, cholate and deoxycholate, two of the most abundant and very closely related physiological bile salts, vary substantially in their destabilizing effects on proteins in vitro and cause protein unfolding of different subsets of proteins in vivo. Our results provide a potential mechanistic explanation for the antimicrobial effects of bile salts, help explain the beneficial effects of bile salt mixtures, and suggest that we have identified a physiological source of protein-unfolding disulfide stress conditions in bacteria.

G-proteins function as molecular switches to power cofactor translocation and confer fidelity in metal trafficking. The G-protein, MMAA, together with MMAB, an adenosyltransferase, orchestrate cofactor delivery and repair of B 12 -dependent human methylmalonyl-CoA mutase (MMUT). The mechanism by which the complex assembles and moves a >1300 Da cargo, or fails in disease, are poorly understood. Herein, we report the crystal structure of the human MMUT-MMAA nano-assembly, which reveals a dramatic 180° rotation of the B 12 domain, exposing it to solvent. The complex, stabilized by MMAA wedging between two MMUT domains, leads to ordering of the switch I and III loops, revealing the molecular basis of mutase-dependent GTPase activation. The structure explains the biochemical penalties incurred by methylmalonic aciduria-causing mutations that reside at the MMAA-MMUT interfaces we identify here. B 12 -dependent human methylmalonly-CoA mutase (MMUT) requires the chaperone MMAA. The authors report the crystal structure of MMUT-MMAA, which shows a MMAA-driven conformational change in MMUT involved n B 12 loading and repair and helps explain the effects of disease-causing MMAA-MMUT interface mutations.

The mystery of how the cyanide group in vitamin B₁₂ or cyanocobalamin, discovered 60 years ago, is removed, has been solved by the demonstration that the trafficking chaperone, MMACHC, catalyzes a reductive decyanation reaction. Electrons transferred from NADPH via cytosolic flavoprotein oxidoreductases are used to cleave the cobalt-carbon bond with reductive elimination of the cyanide ligand. The product, cob(II)alamin, is a known substrate for assimilation into the active cofactor forms, methylcobalamin and 5'-deoxyadenosylcobalamin, and is bound in the \"base-off\" state that is needed by the two B₁₂-dependent target enzymes, methionine synthase and methylmalonyl-CoA mutase. Defects in MMACHC represent the most common cause of inborn errors of B₁₂ metabolism, and our results explain the observation that fibroblasts from these patients are poorly responsive to vitamin B₁₂ but show some metabolic correction with aquocobalamin, a cofactor form lacking the cyanide ligand, which is mirrored by patients showing poorer clinical responsiveness to cyano- versus aquocobalamin.

The mechanisms through which dietary restriction enhances health and longevity in diverse species are unclear. The transsulfuration pathway (TSP) is a highly conserved mechanism for metabolizing the sulfur-containing amino acids, methionine and cysteine. Here we show that Drosophila cystathionine β-synthase (dCBS), which catalyzes the rate-determining step in the TSP, is a positive regulator of lifespan in Drosophila and that the pathway is required for the effects of diet restriction on animal physiology and lifespan. dCBS activity was up-regulated in flies exposed to reduced nutrient conditions, and ubiquitous or neuron-specific transgenic overexpression of dCBS enhanced longevity in fully fed animals. Inhibition of the TSP abrogated the changes in lifespan, adiposity, and protein content that normally accompany diet restriction. RNAi-mediated knockdown of dCBS also limited lifespan extension by diet. Diet restriction reduced levels of protein translation in Drosophila, and we show that this is largely caused by increased metabolic commitment of methionine cycle intermediates to transsulfuration. However, dietary supplementation of methionine restored normal levels of protein synthesis to restricted animals without affecting lifespan, indicating that global reductions in translation alone are not required for diet-restriction longevity. Our results indicate a mechanism by which dietary restriction influences physiology and aging.

The sulfhydration of cysteine residues in proteins is an important mechanism involved in diverse biological processes. We have developed a proteomics approach to quantitatively profile the changes of sulfhydrated cysteines in biological systems. Bioinformatics analysis revealed that sulfhydrated cysteines are part of a wide range of biological functions. In pancreatic β cells exposed to endoplasmic reticulum (ER) stress, elevated H2S promotes the sulfhydration of enzymes in energy metabolism and stimulates glycolytic flux. We propose that transcriptional and translational reprogramming by the integrated stress response (ISR) in pancreatic β cells is coupled to metabolic alternations triggered by sulfhydration of key enzymes in intermediary metabolism. Proteins play essential roles in almost every aspect of a cell’s life, and also contribute to the structure and function of body tissues and organs. Cells and tissues adapt to their continuously changing environments by regulating the activity of their proteins. For example, proteins that are not fully active immediately after they are built instead require further ‘posttranslational’ modifications to become active. Amino acids are the building blocks of proteins, and cysteine amino acids are frequent sites of posttranslational modifications because they are particularly chemically reactive. Under certain conditions inside the cell, the sulfur atom in a cysteine can bond with chemical group containing a second sulfur atom plus a hydrogen atom. This process, which is known as sulfhydration, can be triggered by the presence of the gas molecule, hydrogen sulfide (H2S). The levels of hydrogen sulfide are highly regulated in the body, and it has been suggested to play a role in aging, environmental stress and many diseases. However, it is not clear whether sulfhydration plays a major role in disease conditions by modifying protein activity. Efforts to address this question have been limited by a lack of methods that can measure the extent of sulfhydration of proteins. However, Gao et al. have now devised such a method. The approach takes steps to avoid false-positive and false-negative results, and can identify changes in the sulfhydration of cysteines across the entire complement of proteins produced by a cell, tissue or organ. Gao et al. then used this new method to show that a master regulator of transcription (i.e. a protein that regulates the expression of many genes) causes large-scale changes in cysteine sulfhydration. These large-scale changes resulted in the reprogramming of the cell’s energy metabolism, and further experiments showed that hydrogen sulfide accumulation influences sulfhydration, protein activity and signaling pathways. The development of this new method may now lead to additional discoveries into the role of hydrogen sulfide as a signaling molecule.

Human cystathionine beta-synthase (CBS) catalyzes the first irreversible step in the transsulfuration pathway and commits homocysteine to the synthesis of cysteine. Mutations in CBS are the most common cause of severe hereditary hyperhomocysteinemia. A yeast two-hybrid approach to screen for proteins that interact with CBS had previously identified several components of the sumoylation pathway and resulted in the demonstration that CBS is a substrate for sumoylation. In this study, we demonstrate that sumoylation of CBS is enhanced in the presence of human polycomb group protein 2 (hPc2), an interacting partner that was identified in the initial yeast two-hybrid screen. When the substrates for CBS, homocysteine and serine for cystathionine generation and homocysteine and cysteine for H(2)S generation, are added to the sumoylation mixture, they inhibit the sumoylation reaction, but only in the absence of hPc2. Similarly, the product of the CBS reaction, cystathionine, inhibits sumoylation in the absence of hPc2. Sumoylation in turn decreases CBS activity by approximately 28% in the absence of hPc2 and by 70% in its presence. Based on these results, we conclude that hPc2 serves as a SUMO E3 ligase for CBS, increasing the efficiency of sumoylation. We also demonstrate that gamma-cystathionase, the second enzyme in the transsulfuration pathway is a substrate for sumoylation under in vitro conditions. We speculate that the role of this modification may be for nuclear localization of the cysteine-generating pathway under conditions where nuclear glutathione demand is high.