Carmen Gherasim

Decyanation of vitamin B12 by a trafficking chaperone

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 tinker, tailor, soldier in intracellular B12 trafficking.

The recognition of eight discrete genetic complementation groups among patients with inherited cobalamin disorders provided early insights into the complexity of a cofactor-processing pathway that supports only two known B(12)-dependent enzymes in mammals. With the identification of all eight genes now completed, biochemical interrogations of their functions have started and are providing novel insights into a trafficking pathway involving porters that tinker with and tailor the active cofactor forms and editors that ensure the fidelity of the cofactor loading process. The principles of sequestration and escorted delivery of a rare and reactive organometallic cofactor that are emerging from studies on B(12) might be of general relevance to other cofactor trafficking pathways.

Folate regulation of axonal regeneration in the rodent central nervous system through DNA methylation

The folate pathway plays a crucial role in the regeneration and repair of the adult CNS after injury. Here, we have shown in rodents that such repair occurs at least in part through DNA methylation. In animals with combined spinal cord and sciatic nerve injury, folate-mediated CNS axon regeneration was found to depend on injury-related induction of the high-affinity folate receptor 1 (Folr1). The activity of folate was dependent on its activation by the enzyme dihydrofolate reductase (Dhfr) and a functional methylation cycle. The effect of folate on the regeneration of afferent spinal neurons was biphasic and dose dependent and correlated closely over its dose range with global and gene-specific DNA methylation and with expression of both the folate receptor Folr1 and the de novo DNA methyltransferases. These data implicate an epigenetic mechanism in CNS repair. Folic acid and possibly other nontoxic dietary methyl donors may therefore be useful in clinical interventions to promote brain and spinal cord healing. If indeed the benefit of folate is mediated by epigenetic mechanisms that promote endogenous axonal regeneration, this provides possible avenues for new pharmacologic approaches to treating CNS injuries.

Navigating the B12 Road: Assimilation, Delivery, and Disorders of Cobalamin

The reactivity of the cobalt-carbon bond in cobalamins is the key to their chemical versatility, supporting both methyl transfer and isomerization reactions. During evolution of higher eukaryotes that utilize vitamin B12, the high reactivity of the cofactor coupled with its low abundance pressured development of an efficient system for uptake, assimilation, and delivery of the cofactor to client B12-dependent enzymes. Although most proteins suspected to be involved in B12 trafficking were discovered by 2009, the recent identification of a new protein reveals that the quest for elucidating the intracellular B12 highway is still far from complete. Herein, we review the biochemistry of cobalamin trafficking.

A human vitamin B12 trafficking protein uses glutathione transferase activity for processing alkylcobalamins

Pathways for tailoring and processing vitamins into active cofactor forms exist in mammals that are unable to synthesize these cofactors de novo. A prerequisite for intracellular tailoring of alkylcobalamins entering from the circulation is removal of the alkyl group to generate an intermediate that can subsequently be converted into the active cofactor forms. MMACHC, a cytosolic cobalamin trafficking chaperone, has been shown recently to catalyze a reductive decyanation reaction when it encounters cyanocobalamin. In this study, we demonstrate that this versatile protein catalyzes an entirely different chemical reaction with alkylcobalamins using the thiolate of glutathione for nucleophilic displacement to generate cob(I)alamin and the corresponding glutathione thioether. Biologically relevant thiols, e.g. cysteine and homocysteine, cannot substitute for glutathione. The catalytic turnover numbers for the dealkylation of methylcobalamin and 5′-deoxyadenosylcobalamin by MMACHC are 11.7 ± 0.2 and 0.174 ± 0.006 h−1 at 20 °C, respectively. This glutathione transferase activity of MMACHC is reminiscent of the methyltransferase chemistry catalyzed by the vitamin B12-dependent methionine synthase and is impaired in the cblC group of inborn errors of cobalamin disorders.

Reversible heme-dependent regulation of human cystathionine β-synthase by a flavoprotein oxidoreductase

Human CBS is a PLP-dependent enzyme that clears homocysteine, gates the flow of sulfur into glutathione, and contributes to the biogenesis of H(2)S. The presence of a heme cofactor in CBS is enigmatic, and its conversion from the ferric- to ferrous-CO state inhibits enzyme activity. The low heme redox potential (-350 mV) has raised questions about the feasibility of the ferrous-CO state forming under physiological conditions. Herein, we provide the first evidence of reversible inhibition of CBS by CO in the presence of a human flavoprotein and NADPH. These data provide a mechanism for cross talk between two gas-signaling systems, CO and H(2)S, via heme-mediated allosteric regulation of CBS.

Structural Basis of Multifunctionality in a Vitamin B12-processing Enzyme

An early step in the intracellular processing of vitamin B12 involves CblC, which exhibits dual reactivity, catalyzing the reductive decyanation of cyanocobalamin (vitamin B12), and the dealkylation of alkylcobalamins (e.g. methylcobalamin; MeCbl). Insights into how the CblC scaffold supports this chemical dichotomy have been unavailable despite it being the most common locus of patient mutations associated with inherited cobalamin disorders that manifest in both severe homocystinuria and methylmalonic aciduria. Herein, we report structures of human CblC, with and without bound MeCbl, which provide novel biochemical insights into its mechanism of action. Our results reveal that CblC is the most divergent member of the NADPH-dependent flavin reductase family and can use FMN or FAD as a prosthetic group to catalyze reductive decyanation. Furthermore, CblC is the first example of an enzyme with glutathione transferase activity that has a sequence and structure unrelated to the GST superfamily. CblC thus represents an example of evolutionary adaptation of a common structural platform to perform diverse chemistries. The CblC structure allows us to rationalize the biochemical basis of a number of pathological mutations associated with severe clinical phenotypes.

The thiostrepton A tryptophan methyltransferase TsrM catalyses a cob(II)alamin-dependent methyl transfer reaction

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a novel class of natural products including several antibiotics and bacterial toxins. In countless RiPP biosynthetic pathways, cobalamin-dependent radical SAM (B12/rSAM) enzymes play a pivotal role. In the biosynthetic pathway of the antibiotic and anti-cancer agent thiostrepton A, TsrM, a B12/rSAM enzyme, catalyses the transfer of a methyl group to an electrophilic carbon atom of tryptophan. Here we show that methylcob(III)alamin is the probable physiological enzyme cofactor, and cob(II)alamin rather than cob(I)alamin is a key reaction intermediate. Furthermore, we establish that TsrM and a triple-alanine mutant alkylate cob(II)alamin efficiently leading to the synthesis of MeCbl. Exploiting TsrM substrate ambiguity, we demonstrate that TsrM does not catalyse substrate H-atom abstraction like most radical SAM enzymes. Based on these data, we propose an unprecedented radical-based C-methylation mechanism, which further expands the chemical versatility of rSAM enzymes.

Kinetics of Reversible Reductive Carbonylation of Heme in Human Cystathionine β-synthase

Cystathionine β-synthase (CBS) catalyzes the condensation of homocysteine with serine or cysteine to form cystathionine and water or hydrogen sulfide (H2S), respectively. In addition to pyridoxal phosphate, human CBS has a heme cofactor with cysteine and histidine as ligands. While Fe(III)-CBS is inert to exogenous ligands, Fe(II)-CBS can be reversibly inhibited by carbon monoxide (CO) and reoxidized by O2 to yield superoxide radical. In this study, we have examined the kinetics of Fe(II)CO-CBS formation and reoxidation. Reduction of Fe(III)-CBS by dithionite showed a square root dependence on concentration, indicating that the reductant species was the sulfur dioxide radical anion (SO2(•-)) that exists in rapid equilibrium with S2O4(2-). Formation of Fe(II)CO-CBS from Fe(II)-CBS and 1 mM CO occurred with a rate constant of (3.1 ± 0.4) × 10(-3) s(-1) (pH 7.4, 25 °C). The reaction of Fe(III)-CBS with the reduced form of the flavoprotein methionine synthase reductase in the presence of CO and NADPH resulted in its reduction and carbonylation to form Fe(II)CO-CBS. Fe(II)-CBS was formed as an intermediate with a rate constant of (9.3 ± 2.5) × 10(2) M(-1) s(-1). Reoxidation of Fe(II)CO-CBS by O2 was multiphasic. The major phase showed a hyperbolic dependence on O2 concentration. Although H2S is a product of the CBS reaction and a potential heme ligand, we did not find evidence of an effect of exogenous H2S on activity or heme binding. Reversible reduction of CBS by a physiologically relevant oxidoreductase is consistent with a regulatory role for the heme and could constitute a mechanism for cross talk among the CO, H2S, and superoxide signaling pathways.

Nitrite Reductase Activity and Inhibition of H2S Biogenesis by Human Cystathionine ß-Synthase

Nitrite was recognized as a potent vasodilator >130 years and has more recently emerged as an endogenous signaling molecule and modulator of gene expression. Understanding the molecular mechanisms that regulate nitrite metabolism is essential for its use as a potential diagnostic marker as well as therapeutic agent for cardiovascular diseases. In this study, we have identified human cystathionine ß-synthase (CBS) as a new player in nitrite reduction with implications for the nitrite-dependent control of H2S production. This novel activity of CBS exploits the catalytic property of its unusual heme cofactor to reduce nitrite and generate NO. Evidence for the possible physiological relevance of this reaction is provided by the formation of ferrous-nitrosyl (FeII-NO) CBS in the presence of NADPH, the human diflavin methionine synthase reductase (MSR) and nitrite. Formation of FeII-NO CBS via its nitrite reductase activity inhibits CBS, providing an avenue for regulating biogenesis of H2S and cysteine, the limiting reagent for synthesis of glutathione, a major antioxidant. Our results also suggest a possible role for CBS in intracellular NO biogenesis particularly under hypoxic conditions. The participation of a regulatory heme cofactor in CBS in nitrite reduction is unexpected and expands the repertoire of proteins that can liberate NO from the intracellular nitrite pool. Our results reveal a potential molecular mechanism for cross-talk between nitrite, NO and H2S biology.