Dominique Padovani

Relative Contributions of Cystathionine β-Synthase and γ-Cystathionase to H2S Biogenesis via Alternative Trans-sulfuration Reactions

In mammals, the two enzymes in the trans-sulfuration pathway, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), are believed to be chiefly responsible for hydrogen sulfide (H2S) biogenesis. In this study, we report a detailed kinetic analysis of the human and yeast CBS-catalyzed reactions that result in H2S generation. CBS from both organisms shows a marked preference for H2S generation by β-replacement of cysteine by homocysteine. The alternative H2S-generating reactions, i.e. β-elimination of cysteine to generate serine or condensation of 2 mol of cysteine to generate lanthionine, are quantitatively less significant. The kinetic data were employed to simulate the turnover numbers of the various CBS-catalyzed reactions at physiologically relevant substrate concentrations. At equimolar concentrations of CBS and CSE, the simulations predict that H2S production by CBS would account for ∼25–70% of the total H2S generated via the trans-sulfuration pathway depending on the extent of allosteric activation of CBS by S-adenosylmethionine. The relative contribution of CBS to H2S genesis is expected to decrease under hyperhomocysteinemic conditions. CBS is predicted to be virtually the sole source of lanthionine, and CSE, but not CBS, efficiently cleaves lanthionine. The insensitivity of the CBS-catalyzed H2S-generating reactions to the grade of hyperhomocysteinemia is in stark contrast to the responsiveness of CSE and suggests a previously unrecognized role for CSE in intracellular homocysteine management. Finally, our studies reveal that the profligacy of the trans-sulfuration pathway results not only in a multiplicity of H2S-yielding reactions but also yields novel thioether metabolites, thus increasing the complexity of the sulfur metabolome.

H2S Biogenesis by Human Cystathionine γ-Lyase Leads to the Novel Sulfur Metabolites Lanthionine and Homolanthionine and Is Responsive to the Grade of Hyperhomocysteinemia

Although there is a growing recognition of the significance of hydrogen sulfide (H2S) as a biological signaling molecule involved in vascular and nervous system functions, its biogenesis and regulation are poorly understood. It is widely assumed that desulfhydration of cysteine is the major source of H2S in mammals and is catalyzed by the transsulfuration pathway enzymes, cystathionine β-synthase and cystathionine γ-lyase (CSE). In this study, we demonstrate that the profligacy of human CSE results in a variety of reactions that generate H2S from cysteine and homocysteine. The γ-replacement reaction, which condenses two molecules of homocysteine, yields H2S and a novel biomarker, homolanthionine, which has been reported in urine of homocystinuric patients, whereas a β-replacement reaction, which condenses two molecules of cysteine, generates lanthionine. Kinetic simulations at physiologically relevant concentrations of cysteine and homocysteine, reveal that the α,β-elimination of cysteine accounts for ∼70% of H2S generation. However, the relative importance of homocysteine-derived H2S increases progressively with the grade of hyperhomocysteinemia, and under conditions of severely elevated homocysteine (200 μm), the α,γ-elimination and γ-replacement reactions of homocysteine together are predicted to account for ∼90% of H2S generation by CSE. Excessive H2S production in hyperhomocysteinemia may contribute to the associated cardiovascular pathology.

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.

Energetics of interaction between the G-protein chaperone, MeaB, and B12-dependent methylmalonyl-CoA mutase

MeaB is an auxiliary protein that supports the function of the radical B12-dependent enzyme, methylmalonyl-CoA mutase, although its precise role is not understood. Mutations in the human homolog of MeaB, MMAA, lead to methylmalonic aciduria, an inborn error of metabolism that can be fatal. To obtain insights into the function of this recently discovered protein, we have characterized the entropic and enthalpic contributions to \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \({\Delta}G_{{^\circ}}^{\mathrm{assoc}}\) \end{document} for complexation of MeaB (in the presence and absence of nucleotides) with methylmalonyl-CoA mutase (in the presence and absence of cofactor). The dissociation constant for binding of methylmalonyl-CoA mutase and MeaB ranges from 34 ± 4 to 524 ± 66 nm, depending on the combination of nucleotide and mutase form. Holomutase binds MeaB 15-fold more tightly when the nonhydrolyzable GTP analog, GMPPNP, is bound versus GDP. In contrast, the apomutase binds MeaB with similar affinity in the presence of either nucleotide. Our studies reveal that a large structural rearrangement accompanies interaction between these proteins and buries between ∼4000 and 8600Å2 of surface area, depending on the combination of ligands in the active sites of the two proteins. Furthermore, we demonstrate that MeaB binds GTP and GDP with similar affinity (Kd of 7.3 ± 1.9 and 6.2 ± 0.7 μm, respectively at 20 °C) and has low intrinsic GTPase activity (∼0.04 min–1 at 37 °C), which is stimulated ∼100-fold by methylmalonyl-CoA mutase. These studies provide insights into the energetics of interaction between the radical enzyme methylmalonyl-CoA mutase and MeaB, which are discussed.

A G-protein editor gates coenzyme B12 loading and is corrupted in methylmalonic aciduria

The mechanism by which docking fidelity is achieved for the multitude of cofactor-dependent enzymes is poorly understood. In this study, we demonstrate that delivery of coenzyme B12 or 5′-deoxyadenosylcobalamin by adenosyltransferase to methylmalonyl-CoA mutase is gated by a small G protein, MeaB. While the GTP-binding energy is needed for the editing function; that is, to discriminate between active and inactive cofactor forms, the chemical energy of GTP hydrolysis is required for gating cofactor transfer. The G protein chaperone also exerts its editing function during turnover by using the binding energy of GTP to elicit release of inactive cofactor that is occasionally formed during the catalytic cycle of MCM. The physiological relevance of this mechanism is demonstrated by a patient mutation in methylmalonyl-CoA mutase that does not impair the activity of this enzyme per se but corrupts both the fidelity of the cofactor-loading process and the ejection of inactive cofactor that forms occasionally during catalysis. Consequently, cofactor in the incorrect oxidation state gains access to the mutase active site and is not released if generated during catalysis, leading, respectively, to assembly and accumulation of inactive enzyme and resulting in methylmalonic aciduria.

Adenosyltransferase tailors and delivers coenzyme B12.

The reactivity and relative rarity of most cofactors pose challenges for their delivery to target enzymes. Using kinetic analyses, we demonstrate that adenosyltransferase, which catalyzes the final step in the assimilation of coenzyme B12, directly transfers the cofactor to methylmalonyl coenzyme A mutase. The strategy of using the final enzyme in an assimilation pathway for tailoring a cofactor and delivering it to a dependent enzyme may be general for cofactor trafficking.

Crystal Structure and Mutagenesis of the Metallochaperone MeaB INSIGHT INTO THE CAUSES OF METHYLMALONIC ACIDURIA

MeaB is an auxiliary protein that plays a crucial role in the protection and assembly of the B12-dependent enzyme methylmalonyl-CoA mutase. Impairments in the human homologue of MeaB, MMAA, lead to methylmalonic aciduria, an inborn error of metabolism. To explore the role of this metallochaperone, its structure was solved in the nucleotide-free form, as well as in the presence of product, GDP. MeaB is a homodimer, with each subunit containing a central α/β-core G domain that is typical of the GTPase family, as well as α-helical extensions at the N and C termini that are not found in other metalloenzyme chaperone GTPases. The C-terminal extension appears to be essential for nucleotide-independent dimerization, and the N-terminal region is implicated in protein-protein interaction with its partner protein, methylmalonyl-CoA mutase. The structure of MeaB confirms that it is a member of the G3E family of P-loop GTPases, which contains other putative metallochaperones HypB, CooC, and UreG. Interestingly, the so-called switch regions, responsible for signal transduction following GTP hydrolysis, are found at the dimer interface of MeaB instead of being positioned at the surface of the protein where its partner protein methylmalonyl-CoA mutase should bind. This observation suggests a large conformation change of MeaB must occur between the GDP- and GTP-bound forms of this protein. Because of their high sequence homology, the missense mutations in MMAA that result in methylmalonic aciduria have been mapped onto MeaB and, in conjunction with mutagenesis data, provide possible explanations for the pathology of this disease.

Activation of the anaerobic ribonucleotide reductase by S-adenosylmethionine.

In all living organisms, deoxyribonucleotides, the precursors of DNA, are produced by reduction of the corresponding ribonucleotides. The reaction is catalyzed by an enzyme called ribonucleotide reductase (RNR), which is thus absolutely essential for growth and survival. A number of facultative and strict anaerobes depend on a class III RNR, which is characterized by the presence of a catalytically essential and oxygen-sensitive glycyl radical in the active site. The introduction of the radical into the RNR protein is initiated by a second protein (the activase), which is a member of the recently discovered “radicalSAM” enzyme superfamily. The enzymes of the “radical-SAM” family are characterized by a (4Fe 4S) center, which is chelated by the three cysteines of the conserved Cys-X3-Cys-X2-Cys motif and serving for binding, reducing, and cleaving S-adenosylmethionine (SAM) into methionine and a putative 5’-deoxyadenosyl radical (Ado). It is now generally accepted that, in all these systems, a cluster–SAM complex is formed as a reaction intermediate, since such a complex has been directly observed by ENDOR spectroscopy in the cases of pyruvate-formate lyase activase (PFL) and lysine aminomutase (LAM) and by X-ray crystallography in the cases of biotin synthase (BioB), coproporphyrinogen oxidase (HemN), and MoaA, an enzyme involved in the biosynthesis of the Mo cofactor. It is likely that a cluster–SAM complex is also generated in the activase of the RNR as a precursor of the Ado radical. Glycyl radical formation implies radical transfer from one protein (activase) to the other (RNR). In this work, we investigate the question of whether this transfer occurs by direct attack of Ado8 onto the glycyl residue of the RNR active site or through radical relays along a radical-transfer chain connecting the activase to the RNR. For this purpose, we used HYSCORE (Hyperfine Sublevel Correlation) spectroscopy to demonstrate the intermediate formation of a cluster–SAM complex in the activase and label-transfer experiments with RNR preparations

IcmF Is a Fusion between the Radical B12 Enzyme Isobutyryl-CoA Mutase and Its G-protein Chaperone

Coenzyme B12 is used by two highly similar radical enzymes, which catalyze carbon skeleton rearrangements, methylmalonyl-CoA mutase and isobutyryl-CoA mutase (ICM). ICM catalyzes the reversible interconversion of isobutyryl-CoA and n-butyryl-CoA and exists as a heterotetramer. In this study, we have identified >70 bacterial proteins, which represent fusions between the subunits of ICM and a P-loop GTPase and are currently misannotated as methylmalonyl-CoA mutases. We designate this fusion protein as IcmF (isobutyryl-CoA mutase fused). All IcmFs are composed of the following three domains: the N-terminal 5′-deoxyadenosylcobalamin binding region that is homologous to the small subunit of ICM (IcmB), a middle P-loop GTPase domain, and a C-terminal part that is homologous to the large subunit of ICM (IcmA). The P-loop GTPase domain has very high sequence similarity to the Methylobacterium extorquens MeaB, which is a chaperone for methylmalonyl-CoA mutase. We have demonstrated that IcmF is an active ICM by cloning, expressing, and purifying the IcmFs from Geobacillus kaustophilus, Nocardia farcinica, and Burkholderia xenovorans. This finding expands the known distribution of ICM activity well beyond the genus Streptomyces, where it is involved in polyketides biosynthesis, and suggests a role for this enzyme in novel bacterial pathways for amino acid degradation, myxalamid biosynthesis, and acetyl-CoA assimilation.

EFA6 controls Arf1 and Arf6 activation through a negative feedback loop

Significance EFA6, cytohesins, and BRAGs activate Arf GTPases in endocytic events. They carry a plasma membrane-binding PH domain in tandem with their catalytic Sec7 domain, which is autoinhibitory and mediates a positive feedback loop in cytohesins but not in BRAGs, and has an as-yet unknown role in EFA6 regulation. By reconstituting GDP/GTP exchange on membranes, we find that the PH domain of EFA6 is not autoinhibitory, but supports a negative feedback loop. This loop is controlled by interaction of Arf6-GTP with the PH-Ct domains of EFA6 and monitors Arf1 and Arf6 activation differentially. This suggests that EFA6 and cytohesins might be coupled in a mixed negative-positive feedback loop to shape the level and timing of Arf1 and Arf6 activation in endocytosis. Guanine nucleotide exchange factors (GEFs) of the exchange factor for Arf6 (EFA6), brefeldin A-resistant Arf guanine nucleotide exchange factor (BRAG), and cytohesin subfamilies activate small GTPases of the Arf family in endocytic events. These ArfGEFs carry a pleckstrin homology (PH) domain in tandem with their catalytic Sec7 domain, which is autoinhibitory and supports a positive feedback loop in cytohesins but not in BRAGs, and has an as-yet unknown role in EFA6 regulation. In this study, we analyzed how EFA6A is regulated by its PH and C terminus (Ct) domains by reconstituting its GDP/GTP exchange activity on membranes. We found that EFA6 has a previously unappreciated high efficiency toward Arf1 on membranes and that, similar to BRAGs, its PH domain is not autoinhibitory and strongly potentiates nucleotide exchange on anionic liposomes. However, in striking contrast to both cytohesins and BRAGs, EFA6 is regulated by a negative feedback loop, which is mediated by an allosteric interaction of Arf6-GTP with the PH-Ct domain of EFA6 and monitors the activation of Arf1 and Arf6 differentially. These observations reveal that EFA6, BRAG, and cytohesins have unanticipated commonalities associated with divergent regulatory regimes. An important implication is that EFA6 and cytohesins may combine in a mixed negative-positive feedback loop. By allowing EFA6 to sustain a pool of dormant Arf6-GTP, such a circuit would fulfill the absolute requirement of cytohesins for activation by Arf-GTP before amplification of their GEF activity by their positive feedback loop.