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ABSTRACT: Significance. Hydrogen sulfide, produced by the desulfuration of cysteine or homocysteine, functions as a signaling molecule in an array of physiological processes including regulation of vascular tone, the cellular stress response, apoptosis and inflammation. Recent Advances. The low steady-state levels of H2S in mammalian cells have been shown recently to reflect a balance between its synthesis and its clearance. The subversion of enzymes in the cytoplasmic transsulfuration pathway for producing H2S from cysteine and/or homocysteine versus producing cysteine from homocysteine, presents an interesting regulatory problem. Critical Issues. It is not known under what conditions the enzymes operate in the canonical transsulfuration pathway and how their specificity is switched to catalyze the alternative H2S-producing reactions. Similarly, it is not known if and whether the mitochondrial enzymes, which oxidize sulfide and persulfide (or sulfane sulfur), are regulated to increase or decrease H2S or sulfane-sulfur pools. Future Directions. In this review, we focus on the enzymology of H2S homeostasis and discuss H2S based signaling via persulfidation and thionitrous acid.
Antioxidants & Redox Signaling 04/2013; · 8.20 Impact Factor
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ABSTRACT: The reactivity of the cobalt-carbon bond in cobalamins is the key to their chemical versatility, supporting both methyltransfer and isomerization reactions. During evolution of higher eukaryotes that utilize 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. While 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.
Journal of Biological Chemistry 03/2013; · 4.77 Impact Factor
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ABSTRACT: Locked B(12) : 4-Ethylphenylcobalamin, a novel organometallic arylcobalamin, has been synthesized in a radical reaction. This vitamin B(12) antimetabolite features a strong CoC bond, and represents a "locked" form of vitamin B(12) . It may be used in animal studies to induce functional vitamin B(12) deficiency artificially to help clarify still controversial issues related to the pathophysiology of vitamin B(12) deficiency.
Angewandte Chemie International Edition 02/2013; 52(9):2606-10. · 13.45 Impact Factor
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ABSTRACT: Mutations in cobalamin or B12 trafficking genes needed for cofactor assimilation and targeting lead to inborn errors of cobalamin metabolism. The gene corresponding to one of these loci, cblD, affects both the mitochondrial and cytoplasmic pathways for B12 processing. We have demonstrated that fibroblast cell lines from patients with mutations in CblD, can dealkylate exogenously supplied methylcobalamin (MeCbl), an activity catalyzed by the CblC protein, but show imbalanced intracellular partitioning of the cofactor into the MeCbl and 5'-deoxyadenosylcobalamin (AdoCbl) pools. These results confirm that CblD functions downstream of CblC in the cofactor assimilation pathway and that it plays an important role in controlling the traffic of the cofactor between the competing cytoplasmic and mitochondrial routes for MeCbl and AdoCbl synthesis, respectively. In this study, we report the interaction of CblC with four CblD protein variants with variable N-terminal start sites. We demonstrate that a complex between CblC and CblD can be isolated particularly under conditions that permit dealkylation of alkylcobalamin by CblC or in the presence of the corresponding dealkylated and oxidized product, hydroxocobalamin (HOCbl). A weak CblC·CblD complex is also seen in the presence of cyanocobalamin. Formation of the CblC·CblD complex is observed with all four CblD variants tested suggesting that the N-terminal 115 residues missing in the shortest variant are not essential for this interaction. Furthermore, limited proteolysis of the CblD variants indicates the presence of a stable C-terminal domain spanning residues ∼116-296. Our results are consistent with an adapter function for CblD, which in complex with CblC·HOCbl, or possibly the less oxidized CblC·cob(II)alamin, partitions the cofactor between AdoCbl and MeCbl assimilation pathways.
Biochimie 02/2013; · 3.02 Impact Factor
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ABSTRACT: In this chapter, we focus on the biochemistry of non-corrin cobalt and on a subset of corrinoid-containing enzymes. We review the import of cobalt in prokaryotes and discuss two members of the non-corrin cobalt-dependent enzymes, nitrile hydratase and methionine aminopeptidase. Cobalt is best known for its central role in alkylcorrinoid cofactors, where the unique properties of the cobalt-carbon bond are exploited to catalyze chemically challenging biotransformations. We discuss the import of corrinoids and the reactions catalyzed by the acyl-CoA mutases, the -fastest-growing subfamily of adenosylcobalamin (AdoCbl)-dependent enzymes. AdoCbl is used as a radical reservoir to catalyze 1,2 rearrangement reactions. The loading of AdoCbl-dependent enzymes with the correct cofactor form is critically important for their functions and is gated by chaperones that use the chemical energy of GTP hydrolysis to ensure the fidelity of the process. Recent insights into the organization and editing functions of G-protein chaperones in the context of AdoCbl-dependent enzymes that they support, are discussed.
Metal ions in life sciences. 01/2013; 12:333-74.
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ABSTRACT: Hydrogen sulfide (H2S) is a recently described endogenously produced gaseous signaling molecule, which influences various cellular processes in the central nervous system, cardiovascular system and gastrointestinal tract. The biogenesis of H2S involves the cytoplasmic transsulfuration enzymes, cystathionine β-synthase and γ-cystathionase, while its catabolism occurs in the mitochondrion and couples to the energy-yielding electron transfer chain. Low steady-state levels of H2S appear to be primarily controlled by efficient oxygen-dependent catabolism via sulfide quinone oxidoreductase, persulfide dioxygenase (ETHE1), rhodanese and sulfite oxidase. Mutations in the persulfide dioxgenase, i.e. ETHE1, result in ethylmalonic encephalopathy, an inborn error of metabolism. In this study, we report the biochemical characterization and kinetic properties of human persulfide dioxygenase and describe the biochemical penalties associated with two patient mutations, T152I and D196N. Steady-state kinetic analysis reveals that the T152I mutation results in a 3-fold lower activity, which is correlated with a 3-fold lower iron content compared to the wild-type enzyme. The D196N mutation results in a 2-fold higher KM for the substrate, glutathione persulfide.
Journal of Biological Chemistry 11/2012; · 4.77 Impact Factor
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ABSTRACT: Human cystathionine β-synthase (CBS), a novel heme-containing pyridoxal 5'-phosphate (PLP) enzyme, catalyzes the condensation of homocysteine and serine or cysteine to produce cystathionine and H2O or H2S, respectively. The presence of heme in CBS has limited spectrophotometric characterization of reaction intermediates by masking the absorption of the PLP cofactor. In this report, we have employed difference stopped-flow spectroscopy to characterize reaction intermediates formed under catalytic turnover conditions. The reactions of L-serine and L-cysteine with CBS result in the formation of a common aminoacrylate intermediate (kobs = 0.96 ± 0.02 mM-1 s-1 and 0.38 ± 0.01 mM-1 s-1, respectively at 24 C) with concomitant loss of H2O and H2S, and without detectable accumulation of the external aldimine or other intermediates. Homocysteine reacts with the aminoacrylate intermediate with kobs = 40.6 ± 3.8 s-1, and re-forms the internal aldimine. In the reverse direction, CBS reacts with cystathionine forming the aminoacrylate intermediate with a kobs = 0.38 ± 0.01 mM-1 s-1. This study provides the first insights into the pre-steady state kinetic mechanism of human CBS and indicates that the reaction is likely to be limited by a conformational change leading to product release.
Journal of Biological Chemistry 11/2012; · 4.77 Impact Factor
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ABSTRACT: Human CBS is a unique pyridoxal 5'-phosphate (PLP)-dependent enzyme that has a regulatory heme cofactor. Previous studies have demonstrated the importance of R266, a residue at the heme pocket end of α-helix 8, for communication between the heme and PLP sites. In this study, we have examined the role of the conserved T257 and T260 residues, located at the other end of α-helix 8 on the heme electronic environment and on activity. The mutations at the two positions destabilize PLP binding, lead to lower PLP content, and ~2- to ~500-fold lower activity compared to the wild-type enzyme. Activity is unresponsive to PLP supplementation, consistent with the pyridoxine nonresponsive phenotype of the T257M mutation in a homocystinuric patient. The H2S producing activities, also impacted by the mutations, show a different pattern of inhibition compared to the canonical transsulfuration reaction. Interestingly, the mutants exhibit contrasting sensitivities to the allosteric effector, S-adenosylmethionine (AdoMet); while T257M and T257I are inhibited the other mutants are hyperactivated by AdoMet. All mutants showed an increased propensity of the ferrous heme to form an inactive species with a 424 nm Soret peak and exhibited significantly reduced enzyme activity in the ferrous and ferrous-CO states. Our results provide the first evidence for bidirectional transmission of information between the cofactor binding sites, suggest the additional involvement of this region in allosteric communication with the regulatory AdoMet-binding domain and reveal the potential for independent modulation of the canonical transsulfuration versus H2S-generating reactions catalyzed by CBS.
Journal of Biological Chemistry 09/2012; · 4.77 Impact Factor
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ABSTRACT: Alzheimer's disease (AD) is associated with impaired glutamate clearance and depressed Na(+)/K(+) ATPase levels in AD brain that might lead to a cellular ion imbalance. To test this hypothesis, [Na(+)] and [K(+)] were analyzed in postmortem brain samples of 12 normal and 16 AD individuals, and in cerebrospinal fluid (CSF) from AD patients and matched controls. Statistically significant increases in [Na(+)] in frontal (25%) and parietal cortex (20%) and in cerebellar [K(+)] (15%) were observed in AD samples compared to controls. CSF from AD patients and matched controls exhibited no differences, suggesting that tissue ion imbalances reflected changes in the intracellular compartment. Differences in cation concentrations between normal and AD brain samples were modeled by a 2-fold increase in intracellular [Na(+)] and an 8-15% increase in intracellular [K(+)]. Since amyloid beta peptide (Aβ) is an important contributor to AD brain pathology, we assessed how Aβ affects ion homeostasis in primary murine astrocytes, the most abundant cells in brain tissue. We demonstrate that treatment of astrocytes with the Aβ 25-35 peptide increases intracellular levels of Na(+) (~2-3-fold) and K(+) (~1.5-fold), which were associated with reduced levels of Na(+)/K(+) ATPase and the Na(+)-dependent glutamate transporters, GLAST and GLT-1. Similar increases in astrocytic Na(+) and K(+) levels were also caused by Aβ 1-40, but not by Aβ 1-42 treatment. Our study suggests a previously unrecognized impairment in AD brain cell ion homeostasis that might be triggered by Aβ and could significantly affect electrophysiological activity of brain cells, contributing to the pathophysiology of AD.
Biochimica et Biophysica Acta 07/2012; 1822(11):1671-81. · 4.66 Impact Factor
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ABSTRACT: The recent spate of discoveries of novel acyl-CoA mutases has engendered a growing appreciation for the diversity of 5'-deoxyadenosylcobalamin-dependent rearrangement reactions. The prototype of the reaction catalyzed by these enzymes is the 1,2 interchange of a hydrogen atom with a thioester group leading to a change in the degree of carbon skeleton branching. These enzymes are predicted to share common architectural elements: a Rossman fold and a triose phosphate isomerase (TIM)-barrel domain for binding cofactor and substrate, respectively. Within this family, methylmalonyl-CoA mutase (MCM) is the best studied and is the only member found in organisms ranging from bacteria to man. MCM interconverts (2R)-methylmalonyl-CoA and succinyl-CoA. The more recently discovered family members include isobutyryl-CoA mutase (ICM), which interconverts isobutyryl-CoA and n-butyryl-CoA; ethylmalonyl-CoA mutase, which interconverts (2R)-ethylmalonyl-CoA and (2S)-methylsuccinyl-CoA; and 2-hydroxyisobutyryl-CoA mutase, which interconverts 2-hydroxyisobutyryl-CoA and (3S)-hydroxybutyryl-CoA. A variant in which the two subunits of ICM are fused to a G-protein chaperone, IcmF, has been described recently. In addition to its ICM activity, IcmF also catalyzes the interconversion of isovaleryl-CoA and pivalyl-CoA. This review focuses on the involvement of acyl-CoA mutases in central carbon and secondary bacterial metabolism and on their biotechnological potential for applications ranging from bioremediation to stereospecific synthesis of C2-C5 carboxylic acids and alcohols, and for production of potential commodity and specialty chemicals.
Biochemistry 07/2012; 51(31):6039-46. · 3.42 Impact Factor
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ABSTRACT: Hydrogen sulfide (H(2)S) is a signaling molecule, which influences many physiological processes. While H(2)S is produced and degraded in many cell types, the kinetics of its turnover in different tissues has not been reported. In this study, we have assessed the rates of H(2)S production in murine liver, kidney, and brain homogenates at pH 7.4, 37°C, and at physiologically relevant cysteine concentrations. We have also studied the kinetics of H(2)S clearance by liver, kidney, and brain homogenates under aerobic and anaerobic conditions.
We find that the rate of H(2)S production by these tissue homogenates is considerably higher than background rates observed in the absence of exogenous substrates. An exponential decay of H(2)S with time is observed and, as expected, is significantly faster under aerobic conditions. The half-life for H(2)S under aerobic conditions is 2.0, 2.8, and 10.0 min with liver, kidney, and brain homogenate, respectively. Western-blot analysis of the sulfur dioxygenase, ETHE1, involved in H(2)S catabolism, demonstrates higher steady-state protein levels in liver and kidney versus brain.
By combining experimental and simulation approaches, we demonstrate high rates of tissue H(2)S turnover and provide estimates of steady-state H(2)S levels.
Our study reveals that tissues maintain a high metabolic flux of sulfur through H(2)S, providing a rationale for how H(2)S levels can be rapidly regulated.
Antioxidants & Redox Signaling 03/2012; 17(1):22-31. · 8.20 Impact Factor
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ABSTRACT: 5'-Deoxyadenosylcobalamin (AdoCbl)-dependent isomerases catalyze carbon skeleton rearrangements using radical chemistry. We have recently characterized a fusion protein that comprises the two subunits of the AdoCbl-dependent isobutyryl-CoA mutase flanking a G-protein chaperone and named it isobutyryl-CoA mutase fused (IcmF). IcmF catalyzes the interconversion of isobutyryl-CoA and n-butyryl-CoA, whereas GTPase activity is associated with its G-protein domain. In this study, we report a novel activity associated with IcmF, i.e. the interconversion of isovaleryl-CoA and pivalyl-CoA. Kinetic characterization of IcmF yielded the following values: a K(m) for isovaleryl-CoA of 62 ± 8 μM and V(max) of 0.021 ± 0.004 μmol min(-1) mg(-1) at 37 °C. Biochemical experiments show that an IcmF in which the base specificity loop motif NKXD is modified to NKXE catalyzes the hydrolysis of both GTP and ATP. IcmF is susceptible to rapid inactivation during turnover, and GTP conferred modest protection during utilization of isovaleryl-CoA as substrate. Interestingly, there was no protection from inactivation when either isobutyryl-CoA or n-butyryl-CoA was used as substrate. Detailed kinetic analysis indicated that inactivation is associated with loss of the 5'-deoxyadenosine moiety from the active site, precluding reformation of AdoCbl at the end of the turnover cycle. Under aerobic conditions, oxidation of the cob(II)alamin radical in the inactive enzyme results in accumulation of aquacobalamin. Because pivalic acid found in sludge can be used as a carbon source by some bacteria and isovaleryl-CoA is an intermediate in leucine catabolism, our discovery of a new isomerase activity associated with IcmF expands its metabolic potential.
Journal of Biological Chemistry 12/2011; 287(6):3723-32. · 4.77 Impact Factor
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ABSTRACT: It is widely believed that microglia and monocyte-derived macrophages (collectively referred to as central nervous system (CNS) macrophages) cause excitotoxicity in the diseased or injured CNS. This view has evolved mostly from in vitro studies showing that neurotoxic concentrations of glutamate are released from CNS macrophages stimulated with lipopolysaccharide (LPS), a potent inflammogen. We hypothesized that excitotoxic killing by CNS macrophages is more rigorously controlled in vivo, requiring both the activation of the glutamate/cystine antiporter (system x(c)(-)) and an increase in extracellular cystine, the substrate that drives glutamate release. Here, we show that non-traumatic microinjection of low-dose LPS into spinal cord gray matter activates CNS macrophages but without causing overt neuropathology. In contrast, neurotoxic inflammation occurs when LPS and cystine are co-injected. Simultaneous injection of NBQX, an antagonist of AMPA glutamate receptors, reduces the neurotoxic effects of LPS+cystine, implicating glutamate as a mediator of neuronal cell death in this model. Surprisingly, neither LPS nor LPS+cystine adversely affects survival of oligodendrocytes or oligodendrocyte progenitor cells. Ex vivo analyses show that redox balance in microglia and macrophages is controlled by induction of system x(c)(-) and that high GSH:GSSG ratios predict the neurotoxic potential of these cells. Together, these data indicate that modulation of redox balance in CNS macrophages, perhaps through regulating system x(c)(-), could be a novel approach for attenuating injurious neuroinflammatory cascades.
Experimental Neurology 11/2011; 233(1):333-41. · 4.70 Impact Factor
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ABSTRACT: 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.
Proceedings of the National Academy of Sciences 09/2011; 108(40):16831-6. · 9.68 Impact Factor
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ABSTRACT: 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.
Biochemistry 09/2011; 50(39):8261-3. · 3.42 Impact Factor
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Carsten Kriebitzsch,
Lieve Verlinden,
Guy Eelen,
Natasja M van Schoor,
Karin Swart,
Paul Lips,
Mark B Meyer,
J Wesley Pike,
Steven Boonen,
Carsten Carlberg,
Victor Vitvitsky,
Roger Bouillon, Ruma Banerjee,
Annemieke Verstuyf
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ABSTRACT: High homocysteine (HCY) levels are a risk factor for osteoporotic fracture. Furthermore, bone quality and strength are compromised by elevated HCY owing to its negative impact on collagen maturation. HCY is cleared by cystathionine β-synthase (CBS), the first enzyme in the transsulfuration pathway. CBS converts HCY to cystathionine, thereby committing it to cysteine synthesis. A microarray experiment on MC3T3-E1 murine preosteoblasts treated with 1,25-dihydroxyvitamin D(3) [1,25(OH)(2) D(3) ] revealed a cluster of genes including the cbs gene, of which the transcription was rapidly and strongly induced by 1,25(OH)(2) D(3) . Quantitative real-time PCR and Western blot analysis confirmed higher levels of cbs mRNA and protein after 1,25(OH)(2) D(3) treatment in murine and human cells. Moreover, measurement of CBS enzyme activity and quantitative measurements of HCY, cystathionine, and cysteine concentrations were consistent with elevated transsulfuration activity in 1,25(OH)(2) D(3) -treated cells. The importance of a functional vitamin D receptor (VDR) for transcriptional regulation of cbs was shown in primary murine VDR knockout osteoblasts, in which upregulation of cbs in response to 1,25(OH)(2) D(3) was abolished. Chromatin immunoprecipitation on chip and transfection studies revealed a functional vitamin D response element in the second intron of cbs. To further explore the potential clinical relevance of our ex vivo findings, human data from the Longitudinal Aging Study Amsterdam suggested a correlation between vitamin D status [25(OH)D(3) levels] and HCY levels. In conclusion, this study showed that cbs is a primary 1,25(OH)(2) D(3) target gene which renders HCY metabolism responsive to 1,25(OH)(2) D(3).
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 08/2011; 26(12):2991-3000. · 6.04 Impact Factor
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ABSTRACT: The physiological roles of taurine, a product of cysteine degradation and one of the most abundant amino acids in the body, remain elusive. Taurine deficiency leads to heart dysfunction, brain development abnormalities, retinal degradation, and other pathologies. The taurine synthetic pathway is proposed to be incomplete in astrocytes and neurons, and metabolic cooperation between these cell types is reportedly needed to complete the pathway. In this study, we analyzed taurine synthesis capability as reported by incorporation of radioactivity from [(35)S]cysteine into taurine, in primary murine astrocytes and neurons, and in several transformed cell lines (human (SH-SY5Y) and murine (N1E-115) neuroblastoma, human astrocytoma (U-87 MG and 1321 N1), and rat glioma (C6)). Extensive incorporation of radioactivity from [(35)S]cysteine into taurine was observed in rat glioma cells as well as in primary mouse astrocytes and neurons, establishing the presence of an intact taurine synthesis pathway in these cells. Interestingly, exposure of cells to cysteine or cysteamine resulted in elevated intracellular hypotaurine without a corresponding increase in taurine levels, suggesting that oxidation of hypotaurine limits taurine synthesis in cells. Consistent with its role as an organic osmolyte, taurine synthesis was stimulated under hypertonic conditions in neurons.
Journal of Biological Chemistry 07/2011; 286(37):32002-10. · 4.77 Impact Factor
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ABSTRACT: The synthesis of glutathione, a major cellular antioxidant with a critical role in T cell proliferation, is limited by cysteine. In this study, we evaluated the contributions of the x(C)(-) cystine transporter and the transsulfuration pathway to cysteine provision for glutathione synthesis and antioxidant defense in naïve versus activated T cells and in the immortalized T lymphocyte cell line, Jurkat. We show that the x(C)(-) transporter, although absent in naïve T cells, is induced after activation, releasing T cells from their cysteine dependence on antigen-presenting cells. We also demonstrate the existence of an intact transsulfuration pathway in naïve and activated T cells and in Jurkat cells. The flux through the transsulfuration pathway increases in primary but not in transformed T cells in response to oxidative challenge by peroxide. Inhibition of the transsulfuration pathway in both primary and transformed T cells decreases cell viability under oxidative-stress conditions.
Antioxidants & Redox Signaling 07/2011; 15(1):39-47. · 8.20 Impact Factor
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ABSTRACT: ATP-dependent cob(I)alamin adenosyltransferase (ATR) is a bifunctional protein: an enzyme that catalyzes the adenosylation of cob(I)alamin and an escort that delivers the product, adenosylcobalamin (AdoCbl or coenzyme B(12)), to methylmalonyl-CoA mutase (MCM), resulting in holoenzyme formation. Failure to assemble holo-MCM leads to methylmalonic aciduria. We have previously demonstrated that only 2 equiv of AdoCbl bind per homotrimer of ATR and that binding of ATP to the vacant active site triggers ejection of 1 equiv of AdoCbl from an adjacent site. In this study, we have mimicked in the Methylobacterium extorquens ATR, a C-terminal truncation mutation, D180X, described in a patient with methylmalonic aciduria, and characterized the associated biochemical penalties. We demonstrate that while k(cat) and K(M)(Cob(I)) for D180X ATR are only modestly decreased (by 3- and 2-fold, respectively), affinity for the product, AdoCbl, is significantly diminished (400-fold), and the negative cooperativity associated with its binding is lost. We also demonstrate that the D180X mutation corrupts ATP-dependent cofactor ejection, which leads to transfer of AdoCbl from wild-type ATR to MCM. These results suggest that the pathogenicity of the corresponding human truncation mutant results from its inability to sequester AdoCbl for direct transfer to MCM. Instead, cofactor release into solution is predicted to reduce the capacity for holo-MCM formation, leading to disease.
Biochemistry 06/2011; 50(25):5790-8. · 3.42 Impact Factor
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ABSTRACT: An early step in the intracellular processing of vitamin B(12) involves CblC, which exhibits dual reactivity, catalyzing the reductive decyanation of cyanocobalamin (vitamin B(12)), 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.
Journal of Biological Chemistry 06/2011; 286(34):29780-7. · 4.77 Impact Factor
