Dissertations / Theses on the topic 'Methylmalonyl coenzyme A mutase'

Methylmalonic aciduria (MMA) is an autosomal recessive metabolic disorder with an incidence of 1 in 48,000, which may be due to a defect in the mitochondrial homodimeric enzyme methylmalonyl CoA mutase (mut MMA). mut MMA is subdivided into $mut sp circ$ and $mut sp-$ subclasses on the basis of complementation analysis; $mut sp circ$ cell lines have very low incorporation of ($ sp{14}$C) from propionate into acid precipitable material while incorporation in $mut sp-$ cells is increased when cells are incubated in cobalamin. Intragenic complementation was first observed with WG 1130, a $mut sp circ$ fibroblast line with a homozygous R93H mutation, that is capable of complementing MCM activity when fused with some $mut sp circ$ and some $mut sp-$ cells (1). Extensive intragenic complementation in mut MMA was subsequently observed. Fibroblasts cultured from thirteen unrelated patients (6 $mut sp-$, 7 $mut sp circ$) were fused in all possible pairwise combination and MCM activity was assayed in the heterokaryons by measuring the incorporation of ($ sp{14}$C) from propionate into acid precipitable material. Intragenic complementation, indicated by stimulation of ($ sp{14}$C) -propionate incorporation following cell fusion with polyethylene glycol, was observed in fusions involving twelve of the thirteen strains. Of these thirteen strains, mutations have been identified in six; four have a homozygous mutation (WG 1130 (R93H), WG 1511 (H678R), WG 1610 (G717V), WG 1609 (G630E)), and two cell lines are compound heterozygous (WG 1681 (G623R and G703R), WG 1607 (W105R and A377E)); the remainders are yet to be determined. These intragenic complementations will provide information for grouping the mutations in defined domains in order to correlate structure and function of MCM.

Methylmalonyl-CoA mutase (MCM, E.C. 5.4.99.2), a coenzyme B12-dependent enzyme, catalyses the inter conversion of succinyl-CoA and methylmalonyl- CoA. The gene (sbm) encoding this enzyme is found in Escherichia coli (E. coli) at 62.3min on the E. coli chromosome. However, the metabolic role of this enzyme in the organism is not known. This project involves an investigation into this metabolic obscurity. The sbm gene is part of a four gene operon which also includes argK (or ygfD) that codes for a protein kinase catalysing the phosphorylation of two periplasmic binding proteins involved in cationic amino acid transport, ygfG that codes for methylmalonyl-CoA decarboxylase and ygfH that codes for propionyl-CoA: succinyl-CoA transferase. From existing literature we suspect that this operon, including the sbm gene, could be involved in the utilisation of unusual carbon sources such as succinate and propionate. An insertion mutant of the sbm gene created by transposon mediated mutagenesis was used for investigating the role of this gene. The wild type E. coli K12 strain, E. coli TR6524 and the mutant E. coli K12 (sbm::MudJ) were used in this study. Growth of the two strains (E. coli TR6524 and FA1P1) in minimal media with three different concentrations (0.05, 0.5, 5.0μg/mL) of vitamin B12 and in the presence succinate, propionate or glucose as the sole source of carbon, was studied. Growth was typical in media with glucose with no major differences in the growth pattern of the wild type and mutant strain. However, the two strains exhibited a differential growth pattern in media containing succinate, with the wild type growing faster than the mutant, indicating the role of the sbm gene in the utilisation of this carbon source. Growth in media containing propionate as the sole carbon source indicated only marginal differences in the growth pattern of the wild type and mutant strain. This result possibly suggests that the other pathways for propionate utilisation in E. coli compensate for the lack of a functional Sbm protein in the mutant strain. Promoter analysis indicated the presence of a promoter induced by σS, a transcription factor involved in the expression of proteins under stress or stationary phase growth conditions. Reverse transcription polymerase chain reaction (RT-PCR) studies of the genes of the sbm operon (sbm-argK-ygfGygfH) under the same growth conditions were carried out. Densitometric analysis of the PCR products suggested that the transcription level of sbm was higher in E. coli grown in succinate as compared to when grown in glucose and not as much when grown in propionate indicating a transcriptional level control of the sbm gene expression during the utilisation of succinate. RT-PCR studies also indicated a higher level of transcription of the gene in the stationary phase of the culture during the utilisation of succinate. Real time reverse transcription PCR (QPCR) analysis was used for the absolute quantification of the transcription of the genes of the sbm operon. An increase in the mRNA levels corresponding to the sbm, argK and ygfG genes was observed as E. coli TR6524 growth reached stationary phase, in the presence of succinate or propionate as the sole source of carbon as compared to glucose, In contrast, the highest mRNA levels corresponding to the ygfH gene were observed in the early log-phase of growth. This indicated a differential transcriptional level control of the genes within the operon. This study further established the possible role of this operon in the utilisation of succinate and propionate. The MCM enzyme activity measurement in the whole cell extracts of the wild type E. coli K12, grown under the above mentioned conditions, led to the first ever measurement of MCM activity in wild type E. coli. These measurements also revealed a four fold increase of the MCM specific activity in the case of growth in succinate (4.76x10-3U/mg) and a two fold increase for growth in propionate (2.79x10-3U/mg) compared to that observed with growth in glucose (1.37x10-3U/mg), indicating a significant level of involvement of the enzyme in succinate utilisation, and to a lesser extent in propionate utilisation. The proteomic analysis to understand the gene expression pattern of E. coli TR6524 was carried out using cells harvested at the stationary phase. The results showed that growth conditions induced the expression of transport related (HisJ, DppA) and energy generating proteins (PckA, AceF) required by E. coli to cope with the stressful growth conditions. However, Sbm was not identified among the limited protein spots that were analysed. Finally, E. coli K12 sbm gene was successfully cloned into B. cereus SPV leading to the development of a metabolically engineered polyhydroxyalkanoate producing strain of B. cereus. The intention was to provide the bacteria with a natural intracellular source of propionyl-CoA, leading to the production of the P(3HB-co-3HV) copolymer from structurally non related carbon sources like glucose. Hence, this work has initiated investigation into the metabolic role of the sbm gene product in E. coli. In addition, it has also led to the use of this gene product in metabolic engineering applications.

Methylmalonic aciduria is an autosomal recessive metabolic disorder, which may be due to a defect in the methylmalonyl CoA mutase (MCM) apoenzyme. The mut$ sp circ$ mutation is characterized by undetectable enzyme activity in cell extracts, and by the low incorporation of ($ sp{14}$C) propionate in the presence of hydroxocobalamin in culture. A mut$ sp circ$ fibroblast cell line, WG 1681, from an African-American male infant was shown to complement another mut$ sp circ$ cell line, WG 1130. Subsequent cloning and sequencing of cDNA from WG 1681 identified two previously described homozygous polymorphisms: H532R and V671I(1). In addition, compound heterozygosity was observed for two novel changes at highly conserved sites: G623R and G703R. Hybridization of allele specific oligonucleotides to PCR amplified MCM exons from WG 1681 and family members identified a clinically normal mother, sister and half-brother as carriers of the G703R change in cis with both polymorphisms. The putative father was not identified as a carrier of the G623R change. transfection of each change, singly and in cis with both polymorphisms, into GM1673 cells demonstrated a lack of stimulation of ($ sp{14}$C) propionate uptake in the absence and presence of OH-Cbl, in comparison to controls. Co-transfection of each separate mutation with the previously identified R93H mutation of WG 1130 (2) stimulated propionate uptake. These results indicate that G623R and G703R are novel mutations responsible for deficient MCM activity and the mut$ sp circ$ phenotype in WG 1681, and both mutations are independently capable of complementing the R93H mutation of WG 1130.

Methylmalonic aciduria results from defects in the enzyme methylmalonyl-CoA mutase and from defects in the synthesis of the enzyme's cofactor adenosylcobalamin. Two patients who excrete methylmalonic acid have been shown to have a homozygous nonsense mutation in the methylmalonyl-CoA epimerase gene (MCEE). To further understand the causes of methylmalonic acid excretion, the MCEE gene was sequenced in 229 patients who excreted methylmalonic acid for which no cause was known. Mutations were detected in five patients. Fusion of fibroblast lines from two patients with a homozygous nonsense mutation in MCEE did not result in correction of [14C]propionate incorporation toward control values while the defect in these fibroblasts was complemented by mut, cblA, and cblB fibroblasts. Transfection with wild-type MCEE cDNA resulted in correction of the biochemical phenotype in cells from both patients. These experiments support the hypothesis that a defective epimerase enzyme can be a cause of elevated methylmalonic acid excretion.

La vitamine B12 est la plus connue des cobalamines (Cbl). Parmi ces dernières, seules deux formes sont biologiquement actives chez les mammifères : la méthylcobalamine (CH3Cbl) et la 5' désoxyadénosylcobalamine (AdoCbl). La CH3Cbl est le coenzyme de la méthionine synthétase, enzyme cytoplasmique catalysant la méthylation de l'homocystéine en méthionine. L'AdoCbl est le cofacteur d'une enzyme mitochondriale catalysant l'isomérisation du méthylmalonyl coenzyme A (MMCoA) en succinyl coenzyme A (SCoA) : la méthylmalonyl coenzyme A mutase (MCM). Une carence en Cbl peut se traduire par l'apparition d'une myéloneuropathie. Une des hypothèses émises pour tenter d'élucider les mécanismes biochimiques mis en cause implique l'inhibition de la MCM. D'après cette hypothèse, lors d'une déficience en Cbl, le MMCoA et son précurseur, le propionyl coenzyme A, s'accumuleraient et perturberaient la synthèse des acides gras. Il en résulterait la synthèse d'acides gras anormaux, à squelette ramifié et/ou à nombre impair d'atomes de carbones, responsables de modifications de l'intégrité de la myéline et donc de troubles neurologiques. La majorité des auteurs ayant étudié l'effet de la carence en Cbl sur les activités holoenzymatique (sans ajout de Cbl exogène) et totale de la MCM ont mesuré les activïtés enzymatiques dans différents organes d'animaux expérimentalement carencés en Cbl. Leurs résultats sont contradictoires. Nous nous sommes proposé de mesurer les activités totale et holoenzymatique de la MCM d'oligodendrocytes de rat en culture, placés dans un milieu carencé ou non en Cbl. Ce modèle d'étude présente l'avantage de cibler la cellule atteinte au cours de la démyélinisation, à savoir, dans le système nerveux central, l'oligodendrocyte. Il permet en outre de réaliser rapidement une carence en Cbl. [. . . ]

Les acidémies méthylmaloniques (AMM) isolées sont des erreurs innées du métabolisme génétiquement hétérogènes transmises sur le mode autosomique récessif et secondaires au déficit de l'activité enzymatique de la méthylmalonyl-CoA mutase. La mutase est une enzyme intramitochondriale codée par un gène nucléaire qui possède comme coenzyme un dérivé de la vitamine B12 : l'adénosylcobalamine. Après avoir déterminé l'incidence de ces pathologies (1/140 000), nous avons établi le phénotype biochimique d'une cohorte de 63 patients atteints d'AMM isolée dont 60 présentent une forme mut. Le séquencage du gène de la mutase chez ses 60 patients à permis d'identifier 35 nouvelles mutation sur le locus MUT. Nous avons également étudié à l'aide d'outils comme la PCR quantitative ou un modèle d'expression de protéine de fusion, la régulation de l'expression du gène de la mutase par différentes molécules impliquées ans la régulation de l'expression d'enzyme du métabolisme intermédiaire.

Methylmalonic aciduria (MMAuria) most commonly results from a deficiency of methylmalonyl coenzyme A mutase (MCM). Current treatments for MMAuria remain unsatisfactory and research on novel therapies remains a high priority. A lentiviral (LV) vector was developed to treat in vitro and in vivo models of MMAuria. The overall aim of this project was to examine the therapeutic effect of a LV vector that expresses human MCM transgene in MCM knockout fibroblasts and a MMA affected, mut -/- muth2, murine model. In the first study, a self-inactivating LV vector that expressed human MCM, HIV- 1SDmEF1αhMCM, was constructed and transduced into MCM knockout fibroblasts. Normal cells and untransduced MCM knockout fibroblasts served as controls. Real-time PCR showed a high level of vector copy number, 8 ± 2 copies/cell in LV-treated MCM-knockout fibroblasts, resulting in correction of both the MCM enzyme activity and propionate metabolism in MCM-knockout fibroblasts. The HIV-1SDmEF1αhMCM was then delivered intravenously into mut -/- muth2 mice (n=2). Untreated mut -/- muth2 mice (n=2) and normal mice (n=5) were used as controls. Vector was detected at a copy number of 0.19 ± 0.04 copies/cell in liver. Nevertheless, the MCM enzyme analysis showed only a modest restoration of enzyme activity in the treated mice, resulting in a mild reduction of plasma and urine MMA levels in the treated animals. These data suggest success in targeting the liver with the intravenous gene delivery approach. Nevertheless, it was required to improve the human MCM transgene expression in order to enhance the level of restoration of MCM enzyme activity to further reduce the MMA levels. In the second study, a LV vector that expresses a codon-optimised human MCM transgene, HIV-1SDmEF1αmurSigHutMCM, was produced and transduced into MCM-knockout fibroblasts. High levels of vector, 20 ± 0.8 copies/cell, were detected in LV-treated MCM- knockout fibroblasts. Western blot analysis and MCM enzyme activity analysis by HPLC demonstrated a high level of MCM expression in the treated fibroblasts, resulting in the correction of MCM enzyme activity, with the formation of a significant level of succinyl coenzyme A (179 ± 19 nM/min/μg of total cell protein). The HIV-1SDmEF1αmurSigHutMCM was then injected intravenously into mut -/- muth2 mice (n=5). Untreated mut -/- muth2 (n=6) and normal mice (n=6) were used as controls. The HIV-1SDmEF1αmurSigHutMCM-treated mice achieved near-normal weight for sex. The western blot analysis demonstrated significant MCM enzyme expression in the liver of treated mice, with the measurement of high level of enzyme activity (66 ± 21 nM/min/μg of total cell protein). Biochemical analyses demonstrated that the normalization of MCM enzyme activity in the treated group was associated with a reduction in plasma and urine MMA levels. Furthermore, that a significantly lower MMA concentration, 133± 20 μM/g tissue, was measured in the liver compared to the untreated mice, 1003 ± 124 μM/g tissue. These results confirm that HIV-1SDmEF1αmurSigHutMCM provides significant, if incomplete, biochemical correction for the treatment of this disease, suggesting that gene therapy is a potential treatment for MMAuria.
Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2012

碩士
國立臺北科技大學
生物科技研究所
96
Glutamate mutase from Clostridium tetanomorphum is one of a group of adenosylcobalamin(AdoCbl)-dependent mutases which catalyzes the interconversion of L-glutamate and threo-β-methyl-L-aspartate. Glutamate mutase is comprised of two weakly-associating subunits: GlmE(Mr 53,700Da) and MutS(Mr 14,700Da). To further characterize the interactions between glutamate mutase and the coenzyme three different coenzyme B12 analogs, methylcobinamide (MeCbi), adenosylcobinamide (AdoCbi), and adeosylcobinamide-GDP (AdoCbi-GDP), were chemoenzymaticlly synthesized. In this study, a HPLC-based method was used to investigate the enzyme’s kinetic properties. In each case, kcat was decreased by about 2000 fold when AdoCbi or AdoCbi-GDP was used as cofactor.

碩士
國立陽明大學
遺傳學研究所
91
mut type methylmalonic acidemia (mut MMA, MIM 251000) is an autosomal recessive disorder of organic acid metabolism caused by methylmalonyl CoA mutase (MCM, E.C.5.4.99.2; gene symbol: MUT) deficiency. In this study, 13 exons of the MUT gene were PCR amplified and sequenced subsequently to identify the molecular defects of four unrelated Chinese mut MMA patients. Seven MUT gene mutations, namely c.683G>A (R228Q), c.1050C>G (H350Q), c.1106G>A (R369H), c.1280G>A (G427D), c.1741C>T (R581X), IVS9-1G>A and c.1046_1058del (A349delX368), were identified. Three transitions, c.683G>A, c.1106G>A and c.1280G>A, had been reported in other mut MMA patients. The c.1106G>A and c.1280G>A mutations had been proved to be disease-causing mutations by functional analysis previously. The c.1050C>G, c.1741C>T, IVS9-1G>A substitutions and 1046_1058del are novel mutations found in the MUT gene. None of 100 alleles of unrelated normal Chinese was found to have c.1050C>G, c.1741C>T, IVS9-1G>A and 1046_1058del alterations. The c.683G>A, c.1050C>G and c.1741C>T mutations were found to abolish MCM activity by functional study. These data indicated that these three alterations identified in this study might be the disease-causing mutations in mut MMA patients. The allele frequency of c.1280G>A and c.[1630G>T+1631G>A] mutations identified in Chinese mut MMA patients previously were 17% (3/18) and 17% (3/18), respectively. These two mutations were all linked to the 190bp allele of D6S269 microsatellite marker while the 190bp allele of D6S269 was found to be a rare allele in normal Chinese population. The allele frequency of 190bp allele in Chinese mut MMA patient with c.1280G>A or c.[1630G>T+1631G>A] mutations was statistically different from that in the normal Chinese population. These indicated that c.1280G>A and c.[1630G>T+1631G>A] mutations might have founder effects in Chinese mut type MMA patients.