Genetic optimization of the human gut bacterium Phocaeicola vulgatus for enhanced succinate production.

The demand for sustainably produced bulk chemicals is constantly rising. Succinate serves as a fundamental component in various food, chemical, and pharmaceutical products. Succinate can be produced from sustainable raw materials using microbial fermentation and enzyme-based technologies. Bacteroides and Phocaeicola species, widely distributed and prevalent gut commensals, possess enzyme sets for the metabolization of complex plant polysaccharides and synthesize succinate as a fermentative end product. This study employed novel molecular techniques to enhance succinate yields in the natural succinate producer Phocaeicola vulgatus by directing the metabolic carbon flow toward succinate formation. The deletion of the gene encoding the methylmalonyl-CoA mutase (Δmcm, bvu_0309-0310) resulted in a 95% increase in succinate production, as metabolization to propionate was effectively blocked. Furthermore, deletion of genes encoding the lactate dehydrogenase (Δldh, bvu_2499) and the pyruvate:formate lyase (Δpfl, bvu_2880) eliminated the formation of fermentative end products lactate and formate. By overproducing the transketolase (TKT, BVU_2318) in the triple deletion mutant, succinate production increased from 3.9 mmol/g dry weight in the wild type to 10.9 mmol/g dry weight. Overall, succinate yield increased by 180% in the new mutant strain P. vulgatus Δmcm Δldh Δpfl pG106_tkt relative to the parent strain. This approach is a proof of concept, verifying the genetic accessibility of P. vulgatus, and forms the basis for targeted genetic optimization. The increase of efficiency highlights the huge potential of P. vulgatus as a succinate producer with applications in sustainable bioproduction processes. KEY POINTS: • Deleting methylmalonyl-CoA mutase gene in P. vulgatus doubled succinate production • Triple deletion mutant with transketolase overexpression increased succinate yield by 180% • P. vulgatus shows high potential for sustainable bulk chemical production via genetic optimization.

Mutations Affecting Cellular Levels of Cobalamin (Vitamin B12) Confer Tolerance to Bactericidal Antibiotics in Burkholderia cenocepacia.

The Burkholderia cepacia complex (Bcc) consists of opportunistic pathogens known to cause pneumonia in immunocompromised individuals, especially those with cystic fibrosis. Treating Bcc pneumonia is challenging due to the pathogens' high multidrug resistance. Therefore, inhalation therapy with tobramycin powder, which can achieve high antibiotic concentrations in the lungs, is a promising treatment option. In this study, we investigated potential mechanisms that could compromise the effectiveness of tobramycin therapy. By selecting for B. cenocepacia survivors against tobramycin, we identified three spontaneous mutations that disrupt a gene encoding a key enzyme in the biosynthesis of cobalamin (Vitamin B12). This disruption may affect the production of succinyl-CoA by methylmalonyl-CoA mutase, which requires adenosylcobalamin as a cofactor. The depletion of cellular succinyl-CoA may impact the tricarboxylic acid (TCA) cycle, which becomes metabolically overloaded upon exposure to tobramycin. Consequently, the mutants exhibited significantly reduced reactive oxygen species (ROS) production. Both the wild-type and mutants showed tolerance to tobramycin and various other bactericidal antibiotics under microaerobic conditions. This suggests that compromised ROS-mediated killing, due to the impacted TCA cycle, underlies the mutants' tolerance to bactericidal antibiotics. The importance of ROS-mediated killing and the potential emergence of mutants that evade it through the depletion of cobalamin (Vitamin B12) provide valuable insights for developing strategies to enhance antibiotic treatments of Bcc pneumonia.

A Noble Metal Substitution Leads to B12 Cofactor Mimicry by a Rhodibalamin.

In mammals, cobalamin is an essential cofactor that is delivered by a multitude of chaperones in an elaborate trafficking pathway to two client enzymes, methionine synthase and methylmalonyl-CoA mutase (MMUT). Rhodibalamins, the rhodium analogs of cobalamins, have been described as antimetabolites due to their ability to inhibit bacterial growth. In this study, we have examined the reactivity of adenosylrhodibalamin (AdoRhbl) with two key human chaperones, MMACHC (also known as CblC) and adenosyltransferase (MMAB, also known as ATR), and with the human and Mycobacterium tuberculosis MMUT. We demonstrate that while AdoRhbl binds tightly to all four proteins, the Rh-carbon bond is resistant to homolytic (on MMAB and MMUT) as well as heterolytic (on MMACHC) rupture. On the other hand, MMAB catalyzes Rh-carbon bond formation, converting rhodi(I)balamin in the presence of ATP to AdoRhbl. We report the first crystal structure of a rhodibalamin (AdoRhbl) bound to a B12 protein, i.e., MMAB, in the presence of triphosphate, which shows a weakened but intact Rh-carbon bond. The structure provides insights into how MMAB cleaves the corresponding Co-carbon bond in a sacrificial homolytic reaction that purportedly functions as a cofactor sequestration strategy. Collectively, the study demonstrates that while the noble metal substitution of cobalt by rhodium sets up structural mimicry, it compromises chemistry, which could be exploited for targeting human and bacterial B12 chaperones and enzymes.

Clinical outcomes of patients with mut-type methylmalonic acidemia identified through expanded newborn screening in China.

BACKGROUND: Isolated methylmalonic acidemia, an autosomal recessive disorder of propionate metabolism, is usually caused by mutations in the methylmalonyl-CoA mutase gene (mut-type). Because no universal consensus was made on whether mut-type methylmalonic acidemia should be included in newborn screening (NBS), we aimed to compare the outcome of this disorder detected by NBS with that detected clinically and investigate the influence of NBS on the disease course. DESIGN & METHODS: In this study, 168 patients with mut-type methylmalonic acidemia diagnosed by NBS were compared to 210 patients diagnosed after disease onset while NBS was not performed. Clinical data of these patients from 7 metabolic centers in China were analyzed retrospectively, including initial manifestations, biochemical metabolites, the responsiveness of vitamin B12 therapy, and gene variation, to explore different factors on the long-term outcome. RESULTS: By comparison of the clinically-diagnosed patients, NBS-detected patients showed younger age at diagnosis, less incidence of disease onset, better responsiveness of vitamin B12, younger age at start of treatment, lower levels of biochemical features before and after treatment, and better long-term prognosis (P < 0.01). Onset of disease, blood C3/C2 ratio and unresponsiveness of vitamin B12 were more positively associated with poor outcomes of patients whether identified by NBS. Moreover, the factors above as well as older age at start of treatment were positively associated with mortality. CONCLUSIONS: This research highly demonstrated NBS could prevent major disease-related events and allow an earlier treatment initiation. As a key prognostic factor, NBS is beneficial for improving the overall survival of infants with mut-type methylmalonic acidemia.

Clinical and genetic analysis of methylmalonic aciduria in 60 patients from Southern China: a single center retrospective study.

BACKGROUND: Methylmalonic aciduria (MMA) is a group of rare genetic metabolic disorders resulting from defects in methylmalonyl coenzyme A mutase (MCM) or intracellular cobalamin (cbl) metabolism. MMA patients show diverse clinical and genetic features across different subtypes and populations. METHODS: We retrospectively recruited 60 MMA patients from a single center and diagnosed them based on their clinical manifestations and biochemical assays. We then performed genetic analysis to confirm the diagnosis and identify the causal variants. RESULTS: We confirmed the common clinical manifestations of MMA reported previously. We also described four rare MMA cases with unusual symptoms or genetic variants, such as pulmonary hypertension or limb weakness in late-onset patients. We identified 15 MMACHC and 26 MMUT variants in 57 patients, including 6 novel MMUT variants. Two patients had only one MMAA variant each, and one patient had mild MMA due to mitochondrial DNA depletion syndrome caused by a SUCLA2 variant. Among 12 critically ill patients, isolated MMA was associated with higher C3, blood ammonia, and acidosis, while combined MMA was linked to hydrocephalus on skull MRI. MMACHC c.658-660delAAG and MMUT c.1280G > A variants were correlated with more severe phenotypes. CONCLUSIONS: Our study demonstrates the clinical and genotypic heterogeneity of MMA patients and indicates that metabolic screening and genetic analysis are useful tools to identify rare cases.

Bacteroides methylmalonyl-CoA mutase produces propionate that promotes intestinal goblet cell differentiation and homeostasis.

Propionate is a short-chain fatty acid that is generated upon microbiome-mediated fiber fermentation in the intestine. By modulating immune and metabolic pathways, propionate exerts many health benefits. Key bacterial species, such as Bacteroides thetaiotaomicron, generate propionate, but the biochemical pathways and specific functions remain undetermined. We identified a gene operon-encoding methylmalonyl-CoA mutase (MCM) that contributes to propionate biosynthesis in B. thetaiotaomicron. Colonization of germ-free mice with wild-type or MCM-deficient strains as well as in vitro examination demonstrated that MCM-mediated propionate production promotes goblet cell differentiation and mucus-related gene expression. Intestinal organoids lacking the propionate receptor, GPR41, showed reduced goblet cell differentiation upon MCM-mediated propionate production. Furthermore, although wild-type B. thetaiotaomicron alleviated DSS-induced intestinal inflammation, this effect was abolished in mice receiving the MCM-deficient strain but restored upon propionate supplementation. These data emphasize the critical role of MCM-mediated propionate biosynthesis in goblet cell differentiation, offering potential pathways to ameliorate colitis.

Architecture of the human G-protein-methylmalonyl-CoA mutase nanoassembly for B12 delivery and repair.

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 B12-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 B12 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.

Structural insight into G-protein chaperone-mediated maturation of a bacterial adenosylcobalamin-dependent mutase.

G-protein metallochaperones are essential for the proper maturation of numerous metalloenzymes. The G-protein chaperone MMAA in humans (MeaB in bacteria) uses GTP hydrolysis to facilitate the delivery of adenosylcobalamin (AdoCbl) to AdoCbl-dependent methylmalonyl-CoA mutase, an essential metabolic enzyme. This G-protein chaperone also facilitates the removal of damaged cobalamin (Cbl) for repair. Although most chaperones are standalone proteins, isobutyryl-CoA mutase fused (IcmF) has a G-protein domain covalently attached to its target mutase. We previously showed that dimeric MeaB undergoes a 180° rotation to reach a state capable of GTP hydrolysis (an active G-protein state), in which so-called switch III residues of one protomer contact the G-nucleotide of the other protomer. However, it was unclear whether other G-protein chaperones also adopted this conformation. Here, we show that the G-protein domain in a fused system forms a similar active conformation, requiring IcmF oligomerization. IcmF oligomerizes both upon Cbl damage and in the presence of the nonhydrolyzable GTP analog, guanosine-5'-[(ß,γ)-methyleno]triphosphate, forming supramolecular complexes observable by mass photometry and EM. Cryo-EM structural analysis reveals that the second protomer of the G-protein intermolecular dimer props open the mutase active site using residues of switch III as a wedge, allowing for AdoCbl insertion or damaged Cbl removal. With the series of structural snapshots now available, we now describe here the molecular basis of G-protein-assisted AdoCbl-dependent mutase maturation, explaining how GTP binding prepares a mutase for cofactor delivery and how GTP hydrolysis allows the mutase to capture the cofactor.

The impact of metabolic stressors on mitochondrial homeostasis in a renal epithelial cell model of methylmalonic aciduria.

Methylmalonic aciduria (MMA-uria) is caused by deficiency of the mitochondrial enzyme methylmalonyl-CoA mutase (MUT). MUT deficiency hampers energy generation from specific amino acids, odd-chain fatty acids and cholesterol. Chronic kidney disease (CKD) is a well-known long-term complication. We exposed human renal epithelial cells from healthy controls and MMA-uria patients to different culture conditions (normal treatment (NT), high protein (HP) and isoleucine/valine (I/V)) to test the effect of metabolic stressors on renal mitochondrial energy metabolism. Creatinine levels were increased and antioxidant stress defense was severely comprised in MMA-uria cells. Alterations in mitochondrial homeostasis were observed. Changes in tricarboxylic acid cycle metabolites and impaired energy generation from fatty acid oxidation were detected. Methylcitrate as potentially toxic, disease-specific metabolite was increased by HP and I/V load. Mitophagy was disabled in MMA-uria cells, while autophagy was highly active particularly under HP and I/V conditions. Mitochondrial dynamics were shifted towards fission. Sirtuin1, a stress-resistance protein, was down-regulated by HP and I/V exposure in MMA-uria cells. Taken together, both interventions aggravated metabolic fingerprints observed in MMA-uria cells at baseline. The results point to protein toxicity in MMA-uria and lead to a better understanding, how the accumulating, potentially toxic organic acids might trigger CKD.

Biomarkers to predict disease progression and therapeutic response in isolated methylmalonic acidemia.

Methylmalonic Acidemia (MMA) is a heterogenous group of inborn errors of metabolism caused by a defect in the methylmalonyl-CoA mutase (MMUT) enzyme or the synthesis and transport of its cofactor, 5'-deoxy-adenosylcobalamin. It is characterized by life-threatening episodes of ketoacidosis, chronic kidney disease, and other multiorgan complications. Liver transplantation can improve patient stability and survival and thus provides clinical and biochemical benchmarks for the development of hepatocyte-targeted genomic therapies. Data are presented from a US natural history protocol that evaluated subjects with different types of MMA including mut-type (N = 91), cblB-type (15), and cblA-type MMA (17), as well as from an Italian cohort of mut-type (N = 19) and cblB-type MMA (N = 2) subjects, including data before and after organ transplantation in both cohorts. Canonical metabolic markers, such as serum methylmalonic acid and propionylcarnitine, are variable and affected by dietary intake and renal function. We have therefore explored the use of the 1-13 C-propionate oxidation breath test (POBT) to measure metabolic capacity and the changes in circulating proteins to assess mitochondrial dysfunction (fibroblast growth factor 21 [FGF21] and growth differentiation factor 15 [GDF15]) and kidney injury (lipocalin-2 [LCN2]). Biomarker concentrations are higher in patients with the severe mut0 -type and cblB-type MMA, correlate with a decreased POBT, and show a significant response postliver transplant. Additional circulating and imaging markers to assess disease burden are necessary to monitor disease progression. A combination of biomarkers reflecting disease severity and multisystem involvement will be needed to help stratify patients for clinical trials and assess the efficacy of new therapies for MMA.

Identifying and predicting the pathogenic effects of a novel variant inducing severe early onset MMA: a bioinformatics approach.

BACKGROUND: Methylmalonic acidemia (MMA) is a rare metabolic disorder resulting from functional defects in methylmalonyl-CoA mutase. Mutations in the MMAB gene are responsible for the cblB type of vitamin B12-responsive MMA. RESULTS: This study used Whole-exome sequencing (WES), Sanger sequencing, linkage analysis, and in-silico evaluation of the variants' effect on protein structure and function to confirm their pathogenicity in a 2-day-old neonate presenting an early-onset metabolic crisis and death. WES revealed a homozygous missense variant on chromosome 12, the NM_052845.4 (MMAB):c.557G > A, p.Arg186Gln, in exon 7, a highly conserved and hot spot region for pathogenic variants. After being confirmed by Sanger sequencing, the wild-type and mutant proteins' structure and function were modeled and examined using in-silico bioinformatics tools and compared to the variant NM_052845.4 (MMAB):c.556C > T, p.Arg186Trp, a known pathogenic variant at the same position. Comprehensive bioinformatics analysis showed a significant reduction in the stability of variants and changes in protein-protein and ligand-protein interactions. Interestingly, the variant c.557G > A, p.Arg186Gln depicted more variations in the secondary structure and less binding to the ATP and B12 ligands compared to the c.556C > T, p.Arg186Trp, the known pathogenic variant. CONCLUSION: This study succeeded in expanding the variant spectra of the MMAB, forasmuch as the variant c.557G > A, p.Arg186Gln is suggested as a pathogenic variant and the cause of severe MMA and neonatal death. These results benefit the prenatal diagnosis of MMA in the subsequent pregnancies and carrier screening of the family members. Furthermore, as an auxiliary technique, homology modeling and protein structure and function evaluations could provide geneticists with a more accurate interpretation of variants' pathogenicity.

New insights into the pathophysiology of methylmalonic acidemia.

Methylmalonic acidemia (MMA) is a severe inborn error of metabolism that is characterized by pleiotropic metabolic perturbations and multiorgan pathology. Treatment options are limited and non-curative as the underlying causative molecular mechanisms remain unknown. While earlier studies have focused on the potential direct toxicity of metabolites such as methylmalonic and propionic acid as a mechanism to explain disease pathophysiology, new observations have revealed that aberrant acylation, specifically methylmalonylation, is a characteristic feature of MMA. The mitochondrial sirtuin enzyme SIRT5 is capable of recognizing and removing this PTM, however, reduced protein levels of SIRT5 along with other mitochondrial SIRTs 3 and 4 in MMA and potentially reduced function of all three indicates aberrant acylation may require clinical intervention. Therefore, targeting posttranslational modifications may represent a new therapeutic approach to treat MMA and related organic acidemias.

Bivalent molecular mimicry by ADP protects metal redox state and promotes coenzyme B12 repair.

Control over transition metal redox state is essential for metalloprotein function and can be achieved via coordination chemistry and/or sequestration from bulk solvent. Human methylmalonyl-Coenzyme A (CoA) mutase (MCM) catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA using 5'-deoxyadenosylcobalamin (AdoCbl) as a metallocofactor. During catalysis, the occasional escape of the 5'-deoxyadenosine (dAdo) moiety leaves the cob(II)alamin intermediate stranded and prone to hyperoxidation to hydroxocobalamin, which is recalcitrant to repair. In this study, we have identified the use of bivalent molecular mimicry by ADP, coopting the 5'-deoxyadenosine and diphosphate moieties in the cofactor and substrate, respectively, to protect against cob(II)alamin overoxidation on MCM. Crystallographic and electron paramagnetic resonance (EPR) data reveal that ADP exerts control over the metal oxidation state by inducing a conformational change that seals off solvent access, rather than by switching five-coordinate cob(II)alamin to the more air stable four-coordinate state. Subsequent binding of methylmalonyl-CoA (or CoA) promotes cob(II)alamin off-loading from MCM to adenosyltransferase for repair. This study identifies an unconventional strategy for controlling metal redox state by an abundant metabolite to plug active site access, which is key to preserving and recycling a rare, but essential, metal cofactor.

Structure of metallochaperone in complex with the cobalamin-binding domain of its target mutase provides insight into cofactor delivery.

G-protein metallochaperone MeaB in bacteria [methylmalonic aciduria type A (MMAA) in humans] is responsible for facilitating the delivery of adenosylcobalamin (AdoCbl) to methylmalonyl-CoA mutase (MCM), the only AdoCbl-dependent enzyme in humans. Genetic defects in the switch III region of MMAA lead to the genetic disorder methylmalonic aciduria in which the body is unable to process certain lipids. Here, we present a crystal structure of Methylobacterium extorquens MeaB bound to a nonhydrolyzable guanosine triphosphate (GTP) analog guanosine-5'-[(ß,γ)-methyleno]triphosphate (GMPPCP) with the Cbl-binding domain of its target mutase enzyme (MeMCMcbl). This structure provides an explanation for the stimulation of the GTP hydrolyase activity of MeaB afforded by target protein binding. We find that upon MCMcbl association, one protomer of the MeaB dimer rotates ~180°, such that the inactive state of MeaB is converted to an active state in which the nucleotide substrate is now surrounded by catalytic residues. Importantly, it is the switch III region that undergoes the largest change, rearranging to make direct contacts with the terminal phosphate of GMPPCP. These structural data additionally provide insights into the molecular basis by which this metallochaperone contributes to AdoCbl delivery without directly binding the cofactor. Our data suggest a model in which GTP-bound MeaB stabilizes a conformation of MCM that is open for AdoCbl insertion, and GTP hydrolysis, as signaled by switch III residues, allows MCM to close and trap its cofactor. Substitutions of switch III residues destabilize the active state of MeaB through loss of protein:nucleotide and protein:protein interactions at the dimer interface, thus uncoupling GTP hydrolysis from AdoCbl delivery.

The complex machinery of human cobalamin metabolism.

Vitamin B12 (cobalamin, Cbl) is required as a cofactor by two human enzymes, 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR) and methylmalonyl-CoA mutase (MMUT). Within the body, a vast array of transporters, enzymes and chaperones are required for the generation and delivery of these cofactor forms. How they perform these functions is dictated by the structure and interactions of the proteins involved, the molecular bases of which are only now being elucidated. In this review, we highlight recent insights into human Cbl metabolism and address open questions in the field by employing a protein structure and interactome based perspective. We discuss how three very similar proteins-haptocorrin, intrinsic factor and transcobalamin-exploit slight structural differences and unique ligand receptor interactions to effect selective Cbl absorption and internalisation. We describe recent advances in the understanding of how endocytosed Cbl is transported across the lysosomal membrane and the implications of the recently solved ABCD4 structure. We detail how MMACHC and MMADHC cooperate to modify and target cytosolic Cbl to the client enzymes MTR and MMUT using ingenious modifications to an ancient nitroreductase fold, and how MTR and MMUT link with their accessory enzymes to sustainably harness the supernucleophilic potential of Cbl. Finally, we provide an outlook on how future studies may combine structural and interactome based approaches and incorporate knowledge of post-translational modifications to bring further insights.

Integrated multi-omics reveals anaplerotic rewiring in methylmalonyl-CoA mutase deficiency.

Methylmalonic aciduria (MMA) is an inborn error of metabolism with multiple monogenic causes and a poorly understood pathogenesis, leading to the absence of effective causal treatments. Here we employ multi-layered omics profiling combined with biochemical and clinical features of individuals with MMA to reveal a molecular diagnosis for 177 out of 210 (84%) cases, the majority (148) of whom display pathogenic variants in methylmalonyl-CoA mutase (MMUT). Stratification of these data layers by disease severity shows dysregulation of the tricarboxylic acid cycle and its replenishment (anaplerosis) by glutamine. The relevance of these disturbances is evidenced by multi-organ metabolomics of a hemizygous Mmut mouse model as well as through identification of physical interactions between MMUT and glutamine anaplerotic enzymes. Using stable-isotope tracing, we find that treatment with dimethyl-oxoglutarate restores deficient tricarboxylic acid cycling. Our work highlights glutamine anaplerosis as a potential therapeutic intervention point in MMA.

Cellular and computational models reveal environmental and metabolic interactions in MMUT-type methylmalonic aciduria.

Methylmalonyl-coenzyme A (CoA) mutase (MMUT)-type methylmalonic aciduria is a rare inherited metabolic disease caused by the loss of function of the MMUT enzyme. Patients develop symptoms resembling those of primary mitochondrial disorders, but the underlying causes of mitochondrial dysfunction remain unclear. Here, we examined environmental and genetic interactions in MMUT deficiency using a combination of computational modeling and cellular models to decipher pathways interacting with MMUT. Immortalized fibroblast (hTERT BJ5ta) MMUT-KO (MUTKO) clones displayed a mild mitochondrial impairment in standard glucose-based medium, but they did not to show increased reliance on respiratory metabolism nor reduced growth or viability. Consistently, our modeling predicted MUTKO specific growth phenotypes only for lower extracellular glutamine concentrations. Indeed, two of three MMUT-deficient BJ5ta cell lines showed a reduced viability in glutamine-free medium. Further, growth on 183 different carbon and nitrogen substrates identified increased NADH (nicotinamide adenine dinucleotide) metabolism of BJ5ta and HEK293 MUTKO cells compared with controls on purine- and glutamine-based substrates. With this knowledge, our modeling predicted 13 reactions interacting with MMUT that potentiate an effect on growth, primarily those of secondary oxidation of propionyl-CoA, oxidative phosphorylation and oxygen diffusion. Of these, we validated 3-hydroxyisobutytyl-CoA hydrolase (HIBCH) in the secondary propionyl-CoA oxidation pathway. Altogether, these results suggest compensation for the loss of MMUT function by increasing anaplerosis through glutamine or by diverting flux away from MMUT through the secondary propionyl-CoA oxidation pathway, which may have therapeutic relevance.

Q-Flux: A method to assess hepatic mitochondrial succinate dehydrogenase, methylmalonyl-CoA mutase, and glutaminase fluxes in vivo.

The mammalian succinate dehydrogenase (SDH) complex has recently been shown as capable of operating bidirectionally. Here, we develop a method (Q-Flux) capable of measuring absolute rates of both forward (VSDH(F)) and reverse (VSDH(R)) flux through SDH in vivo while also deconvoluting the amount of glucose derived from four discreet carbon sources in the liver. In validation studies, a mitochondrial uncoupler increased net SDH flux by >100% in awake rodents but also increased SDH cycling. During hyperglucagonemia, attenuated pyruvate cycling enhances phosphoenolpyruvate carboxykinase efficiency to drive increased gluconeogenesis, which is complemented by increased glutaminase (GLS) flux, methylmalonyl-CoA mutase (MUT) flux, and glycerol conversion to glucose. During hyperinsulinemic-euglycemic clamp, both pyruvate carboxylase and GLS are suppressed, while VSDH(R) is increased. Unstimulated MUT is a minor anaplerotic reaction but is readily induced by small amounts of propionate, which elicits glucagon-like metabolic rewiring. Taken together, Q-Flux yields a comprehensive picture of hepatic mitochondrial metabolism and should be broadly useful to researchers.

Novel AAV-mediated genome editing therapy improves health and survival in a mouse model of methylmalonic acidemia.

Methylmalonic acidemia (MMA) is an inborn error of metabolism mostly caused by mutations in the mitochondrial methylmalonyl-CoA mutase gene (MMUT). MMA patients suffer from frequent episodes of metabolic decompensation, which can be life threatening. To mimic both the dietary restrictions and metabolic decompensation seen in MMA patients, we developed a novel protein-controlled diet regimen in a Mmut deficient mouse model of MMA and demonstrated the therapeutic benefit of mLB-001, a nuclease-free, promoterless recombinant AAV GeneRideTM vector designed to insert the mouse Mmut into the endogenous albumin locus via homologous recombination. A single intravenous administration of mLB-001 to neonatal or adult MMA mice prevented body weight loss and mortality when challenged with a high protein diet. The edited hepatocytes expressed functional MMUT protein and expanded over time in the Mmut deficient mice, suggesting a selective growth advantage over the diseased cells. In mice with a humanized liver, treatment with a human homolog of mLB-001 resulted in site-specific genome editing and transgene expression in the transplanted human hepatocytes. Taken together, these findings support the development of hLB-001 that is currently in clinical trials in pediatric patients with severe forms of MMA.

Dioxin-elicited decrease in cobalamin redirects propionyl-CoA metabolism to the ß-oxidation-like pathway resulting in acrylyl-CoA conjugate buildup.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant that induces diverse biological and toxic effects, including reprogramming intermediate metabolism, mediated by the aryl hydrocarbon receptor. However, the specific reprogramming effects of TCDD are unclear. Here, we performed targeted LC-MS analysis of hepatic extracts from mice gavaged with TCDD. We detected an increase in S-(2-carboxyethyl)-L-cysteine, a conjugate from the spontaneous reaction between the cysteine sulfhydryl group and highly reactive acrylyl-CoA, an intermediate in the cobalamin (Cbl)-independent ß-oxidation-like metabolism of propionyl-CoA. TCDD repressed genes in both the canonical Cbl-dependent carboxylase and the alternate Cbl-independent ß-oxidation-like pathways as well as inhibited methylmalonyl-CoA mutase (MUT) at lower doses. Moreover, TCDD decreased serum Cbl levels and hepatic cobalt levels while eliciting negligible effects on gene expression associated with Cbl absorption, transport, trafficking, or derivatization to 5'-deoxy-adenosylcobalamin (AdoCbl), the required MUT cofactor. Additionally, TCDD induced the gene encoding aconitate decarboxylase 1 (Acod1), the enzyme responsible for decarboxylation of cis-aconitate to itaconate, and dose-dependently increased itaconate levels in hepatic extracts. Our results indicate MUT inhibition is consistent with itaconate activation to itaconyl-CoA, a MUT suicide inactivator that forms an adduct with adenosylcobalamin. This adduct in turn inhibits MUT activity and reduces Cbl levels. Collectively, these results suggest the decrease in MUT activity is due to Cbl depletion following TCDD treatment, which redirects propionyl-CoA metabolism to the alternate Cbl-independent ß-oxidation-like pathway. The resulting hepatic accumulation of acrylyl-CoA likely contributes to TCDD-elicited hepatotoxicity and the multihit progression of steatosis to steatohepatitis with fibrosis.