Valentin Cracan

Complementation of mitochondrial electron transport chain by manipulation of the NAD+/NADH ratio.

Taking control of cellular NAD+ concentrations Cellular concentrations of the nicotinamide adenine dinucleotide (NAD+) are critical for proper metabolism and are often altered in aging and disease. To enable better understanding of these processes, Titov et al. altered the concentration of NAD+ in particular cellular compartments. They did this through expression of a bacterial enzyme targeted to specific compartments of human cells in culture. Their experiments emphasize the important role of the electron transport chain in redox transfer of electrons to NADH, rather than proton pumping, in mitochondrial pathogenesis. Science, this issue p. 231 The process of borrowing a bacterial enzyme to control nicotinamide adenine dinucleotide concentrations in mammalian cells is discussed. A decline in electron transport chain (ETC) activity is associated with many human diseases. Although diminished mitochondrial adenosine triphosphate production is recognized as a source of pathology, the contribution of the associated reduction in the ratio of the amount of oxidized nicotinamide adenine dinucleotide (NAD+) to that of its reduced form (NADH) is less clear. We used a water-forming NADH oxidase from Lactobacillus brevis (LbNOX) as a genetic tool for inducing a compartment-specific increase of the NAD+/NADH ratio in human cells. We used LbNOX to demonstrate the dependence of key metabolic fluxes, gluconeogenesis, and signaling on the cytosolic or mitochondrial NAD+/NADH ratios. Expression of LbNOX in the cytosol or mitochondria ameliorated proliferative and metabolic defects caused by an impaired ETC. The results underscore the role of reductive stress in mitochondrial pathogenesis and demonstrate the utility of targeted LbNOX for direct, compartment-specific manipulation of redox state.

IGF2BP2/IMP2-Deficient Mice Resist Obesity through Enhanced Translation of Ucp1 mRNA and Other mRNAs Encoding Mitochondrial Proteins

Although variants in the IGF2BP2/IMP2 gene confer risk for type 2 diabetes, IMP2, an RNA binding protein, is not known to regulate metabolism. Imp2(-/-) mice gain less lean mass after weaning and have increased lifespan. Imp2(-/-) mice are highly resistant to diet-induced obesity and fatty liver and display superior glucose tolerance and insulin sensitivity, increased energy expenditure, and better defense of core temperature on cold exposure. Imp2(-/-) brown fat and Imp2(-/-) brown adipocytes differentiated in vitro contain more UCP1 polypeptide than Imp2(+/+) despite similar levels of Ucp1 mRNA; the Imp2(-/-)adipocytes also exhibit greater uncoupled oxygen consumption. IMP2 binds the mRNAs encoding Ucp1 and other mitochondrial components, and most exhibit increased translational efficiency in the absence of IMP2. In vitro IMP2 inhibits translation of mRNAs bearing the Ucp1 untranslated segments. Thus IMP2 limits longevity and regulates nutrient and energy metabolism in the mouse by controlling the translation of its client mRNAs.

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.

Meclizine inhibits mitochondrial respiration through direct targeting of cytosolic phosphoethanolamine metabolism

Background: Previous studies have shown that meclizine inhibits respiration in intact cells, but not in isolated mitochondria, via an unknown mechanism. Results: Meclizine directly inhibits PCYT2 (CTP:phosphoethanolamine cytidylyltransferase). Conclusion: Meclizine attenuates mitochondrial respiration by directly inhibiting the Kennedy pathway of phosphatidylethanolamine biosynthesis. Significance: We identified a novel molecular target of meclizine, an over-the-counter antinausea drug, raising possibilities for new clinical applications. We recently identified meclizine, an over-the-counter drug, as an inhibitor of mitochondrial respiration. Curiously, meclizine blunted respiration in intact cells but not in isolated mitochondria, suggesting an unorthodox mechanism. Using a metabolic profiling approach, we now show that treatment with meclizine leads to a sharp elevation of cellular phosphoethanolamine, an intermediate in the ethanolamine branch of the Kennedy pathway of phosphatidylethanolamine biosynthesis. Metabolic labeling and in vitro enzyme assays confirmed direct inhibition of the cytosolic enzyme CTP:phosphoethanolamine cytidylyltransferase (PCYT2). Inhibition of PCYT2 by meclizine led to rapid accumulation of its substrate, phosphoethanolamine, which is itself an inhibitor of mitochondrial respiration. Our work identifies the first pharmacologic inhibitor of the Kennedy pathway, demonstrates that its biosynthetic intermediate is an endogenous inhibitor of respiration, and provides key mechanistic insights that may facilitate repurposing meclizine for disorders of energy metabolism.

Novel Coenzyme B12-dependent Interconversion of Isovaleryl-CoA and Pivalyl-CoA

Background: IcmF is a fusion between a coenzyme B12-dependent isobutyryl-CoA/n-butyryl-CoA isomerase and a G-protein chaperone. Results: IcmF also isomerizes isovaleryl-CoA to pivalyl-CoA and is partially protected from inactivation in the presence of GTP. Conclusion: The isovaleryl-CoA mutase activity of IcmF might be important in leucine catabolism where isovaleric acid is an intermediate. Significance: IcmF might be critical for microbial bioremediation of the anthropogenic compound pivalic acid. 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 Km for isovaleryl-CoA of 62 ± 8 μm and Vmax 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.

Visualization of a radical B12 enzyme with its G-protein chaperone

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.

Cobalt and Corrinoid Transport and Biochemistry

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.

A genetically encoded tool for manipulation of NADP + /NADPH in living cells

NADH and NADPH are redox coenzymes broadly required for energy metabolism, biosynthesis and detoxification. Despite detailed knowledge of specific enzymes and pathways that utilize these coenzymes, a holistic understanding of the regulation and compartmentalization of NADH and NADPH-dependent pathways is lacking, in part because of a lack of tools with which to investigate them in living cells. We previously reported the use of the naturally occurring Lactobacillus brevis H2O-forming NADH oxidase (LbNOX) as a genetic tool for manipulation of the NAD+/NADH ratio in human cells. Here we present TPNOX (triphosphopyridine nucleotide oxidase), a rationally designed and engineered mutant of LbNOX that is strictly specific towards NADPH. We characterize the effects of TPNOX expression on cellular metabolism and use it in combination with LbNOX to show how the redox states of mitochondrial NADPH and NADH pools are connected.

Engineered and Native Coenzyme B12-dependent Isovaleryl-CoA/Pivalyl-CoA Mutase

Background: IcmF exhibits low isovaleryl-CoA/pivalyl-CoA mutase (PCM) activity. Results: IcmF mutants designed to enhance PCM activity were susceptible to inactivation prompting a bioinformatics search for a “bona fide” PCM. Conclusion: A B12-dependent PCM was identified, cloned, and expressed and exhibited PCM activity. Significance: The newly discovered PCM could be useful in bioremediation and biosynthetic reactions. Adenosylcobalamin-dependent isomerases catalyze carbon skeleton rearrangements using radical chemistry. We have recently demonstrated that an isobutyryl-CoA mutase variant, IcmF, a member of this enzyme family that catalyzes the interconversion of isobutyryl-CoA and n-butyryl-CoA also catalyzes the interconversion between isovaleryl-CoA and pivalyl-CoA, albeit with low efficiency and high susceptibility to inactivation. Given the biotechnological potential of the isovaleryl-CoA/pivalyl-CoA mutase (PCM) reaction, we initially attempted to engineer IcmF to be a more proficient PCM by targeting two active site residues predicted based on sequence alignments and crystal structures, to be key to substrate selectivity. Of the eight mutants tested, the F598A mutation was the most robust, resulting in an ∼17-fold increase in the catalytic efficiency of the PCM activity and a concomitant ∼240-fold decrease in the isobutyryl-CoA mutase activity compared with wild-type IcmF. Hence, mutation of a single residue in IcmF tuned substrate specificity yielding an ∼4000-fold increase in the specificity for an unnatural substrate. However, the F598A mutant was even more susceptible to inactivation than wild-type IcmF. To circumvent this limitation, we used bioinformatics analysis to identify an authentic PCM in genomic databases. Cloning and expression of the putative AdoCbl-dependent PCM with an α2β2 heterotetrameric organization similar to that of isobutyryl-CoA mutase and a recently characterized archaeal methylmalonyl-CoA mutase, allowed demonstration of its robust PCM activity. To simplify kinetic analysis and handling, a variant PCM-F was generated in which the αβ subunits were fused into a single polypeptide via a short 11-amino acid linker. The fusion protein, PCM-F, retained high PCM activity and like PCM, was resistant to inactivation. Neither PCM nor PCM-F displayed detectable isobutyryl-CoA mutase activity, demonstrating that PCM represents a novel 5′-deoxyadenosylcobalamin-dependent acyl-CoA mutase. The newly discovered PCM and the derivative PCM-F, have potential applications in bioremediation of pivalic acid found in sludge, in stereospecific synthesis of C5 carboxylic acids and alcohols, and in the production of potential commodity and specialty chemicals.

Structural Basis for Substrate Specificity in Adenosylcobalamin-dependent Isobutyryl-CoA Mutase and Related Acyl-CoA Mutases

Background: Acyl-CoA mutases catalyze radical-based carbon skeleton rearrangements. Results: Crystal structures of isobutyryl-CoA mutase in complex with four different substrates reveal active site architecture and determinants of substrate specificity. Conclusion: Identification of specificity-determining residues allows for prediction of new acyl-CoA mutase activities. Significance: Improved understanding of acyl-CoA mutase substrate specificity is critical for biotechnological and engineering applications. Acyl-CoA mutases are a growing class of adenosylcobalamin-dependent radical enzymes that perform challenging carbon skeleton rearrangements in primary and secondary metabolism. Members of this class of enzymes must precisely control substrate positioning to prevent oxidative interception of radical intermediates during catalysis. Our understanding of substrate specificity and catalysis in acyl-CoA mutases, however, is incomplete. Here, we present crystal structures of IcmF, a natural fusion protein variant of isobutyryl-CoA mutase, in complex with the adenosylcobalamin cofactor and four different acyl-CoA substrates. These structures demonstrate how the active site is designed to accommodate the aliphatic acyl chains of each substrate. The structures suggest that a conformational change of the 5′-deoxyadenosyl group from C2′-endo to C3′-endo could contribute to initiation of catalysis. Furthermore, detailed bioinformatic analyses guided by our structural findings identify critical determinants of acyl-CoA mutase substrate specificity and predict new acyl-CoA mutase-catalyzed reactions. These results expand our understanding of the substrate specificity and the catalytic scope of acyl-CoA mutases and could benefit engineering efforts for biotechnological applications ranging from production of biofuels and commercial products to hydrocarbon remediation.