Human biotin-containing subunit of 3-methylcrotonyl-CoA carboxylase gene (MCCA): cDNA sequence, genomic organization, localization to chromosomal band 3q27, and expression

3-Methylcrotonyl-CoA carboxylase (MCCase; EC 6.4.1.4) is a mitochondrial biotin enzyme and plays an essential role in the catabolism of leucine and isovalerate in animals, bacterial species, and plants. MCCase consists of two subunits, those that are biotin-containing and non-biotin-containing. The genes responsible for these subunits have been isolated in soybean, Arabidopsis thaliana, and tomatoes, but not in mammals. In humans, MCCase deficiency has been thought to be a rare metabolic disease, but the number of patients with MCCase deficiency appears to be increasing with a wide range of clinical presentations, some that result in a lethal condition and others that are asymptomatic. In this report, we have isolated and carried out chromosomal mapping of the gene for the biotin-containing subunit (A subunit) of the human MCCase gene, MCCA. The cDNA predicts an open reading frame coding for a 725-amino-acid protein with mitochondrial signal peptide, biotin carboxylase, and biotin-carrier domains. The gene is composed of at least 19 exons and covers more than 70 kb of sequence on band q27 of chromosome 3. MCCA was abundantly expressed in mitochondria-rich organs, such as the heart, skeletal muscles, kidney, and liver. In exon 13, we observed a His/Pro polymorphism at codon 464 (an A to C transition at nucleotide position 1391 in the cDNA sequence). Then, we determined the DNA sequences of the 5' untranslated region and entire coding regions in two patients with MCCase deficiency, but no sequence substitution was detected, suggesting that the gene mutations might be in the non-biotin-containing subunit (B subunit) gene, MCCB, in these patients.     

3-Methylcrotonyl-Coenzyme A Carboxylase Is a Component of the Mitochondrial Leucine Catabolic Pathway in Plants

3-Methylcrotonyl-coenzyme A carboxylase (MCCase) is a mitochondrial biotin-containing enzyme whose metabolic function is not well understood in plants. In soybean (Glycine max) seedlings the organ-specific and developmentally induced changes in MCCase expression are regulated by mechanisms that control the accumulation of MCCase mRNA and the activity of the enzyme. During soybean cotyledon development, when seed-storage proteins are degraded, leucine (Leu) accumulation peaks transiently at 8 d after planting. The coincidence between peak MCCase expression and the decline in Leu content provides correlative evidence that MCCase is involved in the mitochondrial catabolism of Leu. Direct evidence for this conclusion was obtained from radiotracer metabolic studies using extracts from isolated mitochondria. These experiments traced the metabolic fate of [U-¹⁴C]Leu and NaH¹⁴CO₃, the latter of which was incorporated into methylglutaconyl-coenzyme A (CoA) via MCCase. These studies directly demonstrate that plant mitochondria can catabolize Leu via the following scheme: Leu → α-ketoisocaproate → isovaleryl-CoA → 3-methylcrotonyl-CoA → 3-methylglutaconyl-CoA → 3-hydroxy-3-methylglutaryl-CoA → acetoacetate + acetyl-CoA. These findings demonstrate for the first time, to our knowledge, that the enzymes responsible for Leu catabolism are present in plant mitochondria. We conclude that a primary metabolic role of MCCase in plants is the catabolism of Leu.     

Disruptions in valine degradation affect seed development and germination in Arabidopsis

We have functionally characterized the role of two putative mitochondrial enzymes in valine degradation using insertional mutants. Prior to this study, the relationship between branched‐chain amino acid degradation (named for leucine, valine and isoleucine) and seed development was limited to leucine catabolism. Using a reverse genetics approach, we show that disruptions in the mitochondrial valine degradation pathway affect seed development and germination in Arabidopsis thaliana. A null mutant of 3‐hydroxyisobutyryl‐CoA hydrolase (CHY4, At4g31810) resulted in an embryo lethal phenotype, while a null mutant of methylmalonate semialdehyde dehydrogenase (MMSD, At2g14170) resulted in seeds with wrinkled coats, decreased storage reserves, elevated valine and leucine, and reduced germination rates. These data highlight the unique contributions CHY4 and MMSD make to the overall growth and viability of plants. It also increases our knowledge of the role branched‐chain amino acid catabolism plays in seed development and amino acid homeostasis.     

The molecular basis of 3-methylcrotonylglycinuria, a disorder of leucine catabolism

3-Methylcrotonylglycinuria is an inborn error of leucine catabolism and has a recessive pattern of inheritance that results from the deficiency of 3-methylcrotonyl-CoA carboxylase (MCC). The introduction of tandem mass spectrometry in newborn screening has revealed an unexpectedly high incidence of this disorder, which, in certain areas, appears to be the most frequent organic aciduria. MCC, an heteromeric enzyme consisting of alpha (biotin-containing) and beta subunits, is the only one of the four biotin-dependent carboxylases known in humans that has genes that have not yet been characterized, precluding molecular studies of this disease. Here we report the characterization, at the genomic level and at the cDNA level, of both the MCCA gene and the MCCB gene, encoding the MCC alpha and MCC beta subunits, respectively. The 19-exon MCCA gene maps to 3q25-27 and encodes a 725-residue protein with a biotin attachment site; the 17-exon MCCB gene maps to 5q12-q13 and encodes a 563-residue polypeptide. We show that disease-causing mutations can be classified into two complementation groups, denoted "CGA" and "CGB." We detected two MCCA missense mutations in CGA patients, one of which leads to absence of biotinylated MCC alpha. Two MCCB missense mutations and one splicing defect mutation leading to early MCC beta truncation were found in CGB patients. A fourth MCCB mutation also leading to early MCC beta truncation was found in two nonclassified patients. A fungal model carrying an mccA null allele has been constructed and was used to demonstrate, in vivo, the involvement of MCC in leucine catabolism. These results establish that 3-methylcrotonylglycinuria results from loss-of-function mutations in the genes encoding the alpha and beta subunits of MCC and complete the genetic characterization of the four human biotin-dependent carboxylases.     

Isolated 3-methylcrotonyl-CoA carboxylase deficiency: evidence for an allele-specific dominant negative effect and responsiveness to biotin therapy

Deficiency of 3-methylcrotonyl-CoA carboxylase (MCC) results in elevated excretion of 3-methylcrotonylglycine (3-MCG) and 3-hydroxyisovaleric acid (3-HIVA). MCC is a heteromeric mitochondrial enzyme comprising biotin-containing alpha subunits and smaller beta subunits, encoded by MCCA and MCCB, respectively. Mutations in these genes cause isolated MCC deficiency, an autosomal recessive disorder with a variable phenotype that ranges from severe neonatal to asymptomatic adult forms. No reported patients have responded to biotin therapy. Here, we describe two patients with a biochemical and, in one case, clinical phenotype of MCC deficiency, both of whom were responsive to biotin. The first patient presented at 3 months with seizures and progressive psychomotor retardation. Metabolic investigation at 2 years revealed elevated excretion of 3-MCG and 3-HIVA, suggesting MCC deficiency. High-dose biotin therapy was associated with a dramatic reduction in seizures, normalization of the electroencephalogram, and correction of the organic aciduria, within 4 weeks. MCC activity in fibroblasts was 25% of normal levels. The second patient, a newborn detected by tandem-mass-spectrometry newborn screening, displayed the same biochemical phenotype and remained asymptomatic with biotin up to the age of 18 months. In both patients, sequence analysis of the complete open reading frames of MCCA and MCCB revealed heterozygosity for MCCA-R385S and for the known polymorphic variant MCCA-P464H but revealed no other coding alterations. MCCA-R385S is unusual, in that it has a normal amount of MCC alpha protein but confers no MCC activity. We show that MCCA-R385S, but not other MCCA missense alleles, reduces the MCC activity of cotransfected MCCA-wild-type allele. Our results suggest that MCCA-R385S is a dominant negative allele and is biotin responsive in vivo.     

Development of glutathione-deficient embryos in Arabidopsis is influenced by the maternal level of glutathione

Glutathione (GSH) biosynthesis-deficient gsh1 and gsh2 null mutants of Arabidopsis thaliana have late embryonic-lethal and early seedling-lethal phenotypes, respectively, when segregating from a phenotypically wild-type parent plant, indicating that GSH is required for seed maturation and during germination. In this study, we show that gsh2 embryos generated in a partially GSH-deficient parent plant, homozygous for either the cad2 mutation in the GSH1 gene or homozygous for mutations in CLT1, CLT2 and CLT3 encoding plastid thiol transporters, abort early in embryogenesis. In contrast, individuals homozygous for the same combinations of mutations but segregating from heterozygous, phenotypically wild-type parents exhibit the parental gsh2 seedling-lethal phenotype. Similarly, homozygous gsh1 embryos generated in a gsh1/cad2 partially GSH-deficient parent plant abort early in development. These observations indicate that the development of gsh1 and gsh2 embryos to a late stage is dependent on the level of GSH in the maternal plant.     

Molecular characterization of the non-biotin-containing subunit of 3-methylcrotonyl-CoA carboxylase

The biotin enzyme, 3-methylcrotonyl-CoA carboxylase (MCCase) (3-methylcrotonyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1. 4), catalyzes a pivotal reaction required for both leucine catabolism and isoprenoid metabolism. MCCase is a heteromeric enzyme composed of biotin-containing (MCC-A) and non-biotin-containing (MCC-B) subunits. Although the sequence of the MCC-A subunit was previously determined, the primary structure of the MCC-B subunit is unknown. Based upon sequences of biotin enzymes that use substrates structurally related to 3-methylcrotonyl-CoA, we isolated the MCC-B cDNA and gene of Arabidopsis. Antibodies directed against the bacterially produced recombinant protein encoded by the MCC-B cDNA react solely with the MCC-B subunit of the purified MCCase and inhibit MCCase activity. The primary structure of the MCC-B subunit shows the highest similarity to carboxyltransferase domains of biotin enzymes that use methyl-branched thiol esters as substrate or products. The single copy MCC-B gene of Arabidopsis is interrupted by nine introns. MCC-A and MCC-B mRNAs accumulate in all cell types and organs, with the highest accumulation occurring in rapidly growing and metabolically active tissues. In addition, these two mRNAs accumulate coordinately in an approximately equal molar ratio, and they each account for between 0.01 and 0.1 mol % of cellular mRNA. The sequence of the Arabidopsis MCC-B gene has enabled the identification of animal paralogous MCC-B cDNAs and genes, which may have an impact on the molecular understanding of the lethal inherited metabolic disorder methylcrotonylglyciuria.     

Substrate specificity of the 3-methylcrotonyl coenzyme A (CoA) and geranyl-CoA carboxylases from Pseudomonas aeruginosa

Biotin-containing 3-methylcrotonyl coenzyme A (MC-CoA) carboxylase (MCCase) and geranyl-CoA (G-CoA) carboxylase (GCCase) from Pseudomonas aeruginosa were expressed as His-tagged recombinant proteins in Escherichia coli. Both native and recombinant MCCase and GCCase showed pH and temperature optima of 8.5 and 37 degrees C. The apparent K(0.5) (affinity constant for non-Michaelis-Menten kinetics behavior) values of MCCase for MC-CoA, ATP, and bicarbonate were 9.8 microM, 13 microM, and 0.8 microM, respectively. MCCase activity showed sigmoidal kinetics for all the substrates and did not carboxylate G-CoA. In contrast, GCCase catalyzed the carboxylation of both G-CoA and MC-CoA. GCCase also showed sigmoidal kinetic behavior for G-CoA and bicarbonate but showed Michaelis-Menten kinetics for MC-CoA and the cosubstrate ATP. The apparent K(0.5) values of GCCase were 8.8 microM and 1.2 microM for G-CoA and bicarbonate, respectively, and the apparent K(m) values of GCCase were 10 microM for ATP and 14 microM for MC-CoA. The catalytic efficiencies of GCCase for G-CoA and MC-CoA were 56 and 22, respectively, indicating that G-CoA is preferred over MC-CoA as a substrate. The enzymatic properties of GCCase suggest that it may substitute for MCCase in leucine catabolism and that both the MCCase and GCCase enzymes play important roles in the leucine and acyclic terpene catabolic pathways.     

Purification and characterization of a <Emphasis Type="Italic">Galactomyces reessii</Emphasis> hydratase that converts 3-methylcrotonic acid to 3-hydroxy-3-methylbutyric acid

Cell free extracts of Galactomyces reessii contain a hydratase as the key enzyme for the transformation of 3-methylcrotonic acid to 3-hydroxy-3-methylbutyric acid. Highest levels of hydratase activity were obtained during growth on isovaleric acid. The enzyme, an enoyl CoA hydratase, was purified 147-fold by precipitation with ammonium sulphate and successive chromatography over columns of DE-52, Blue Sepharose CL-6B and Sephacryl S-200. During purification, hydratase activity was measured spectrophotometrically (OD change at 263 nm) for 3-methylcrotonyl CoA and crotonyl CoA as substrates. The enzyme displayed highest activity with crotonyl CoA with a K _cat of 1,050,000 min⁻¹. The ratio of crotonyl CoA to 3-methylcrotonyl CoA activities was constant (20:1) during all steps of purification. The K _cat for crotonyl CoA was also about 20 times greater than the K _cat for 3-methylcrotonyl CoA (51,700 min⁻¹). The enzyme had pH and temperature optima at 7.0 and 35°C, a native M _r of 260±4.5 kDa and a subunit M _r of 65 kDa, suggesting that the enzyme was a homotetramer. The pI of the purified hydratase was 5.5, and the N-terminal amino acid sequence was VPEGYAEDLLKGKMMRFFDS. Hydratase activity for 3-methylcrotonyl CoA was competitively inhibited by acetyl CoA, propionyl CoA and acetoacetyl CoA. Journal of Industrial Microbiology & Biotechnology (2002) 28, 81–87 DOI: 10.1038/sj/jim/7000215 Received 27 June 2001/ Accepted in revised form 17 September 2001     

The mitochondrial isovaleryl-coenzyme a dehydrogenase of arabidopsis oxidizes intermediates of leucine and valine catabolism

We recently identified a cDNA encoding a putative isovaleryl-coenzyme A (CoA) dehydrogenase in Arabidopsis (AtIVD). In animals, this homotetrameric enzyme is located in mitochondria and catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA as an intermediate step in the leucine (Leu) catabolic pathway. Expression of AtIVD:smGFP4 fusion proteins in tobacco (Nicotiana tabacum) protoplasts and biochemical studies now demonstrate the in vivo import of the plant isovaleryl-CoA dehydrogenase (IVD) into mitochondria and the enzyme in the matrix of these organelles. Two-dimensional separation of mitochondrial proteins by blue native and SDS-PAGE and size determination of the native and overexpressed proteins suggest homodimers to be the dominant form of the plant IVD. Northern-blot hybridization and studies in transgenic Arabidopsis plants expressing Ativd promoter:gus constructs reveal strong expression of this gene in seedlings and young plants grown in the absence of sucrose, whereas promoter activity in almost all tissues is strongly inhibited by exogeneously added sucrose. Substrate specificity tests with AtIVD expressed in Escherichia coli indicate a strong preference toward isovaleryl-CoA but surprisingly also show considerable activity with isobutyryl-CoA. This strongly indicates a commitment of the enzyme in Leu catabolism, but the activity observed with isobutyryl-CoA also suggests a parallel involvement of the enzyme in the dehydrogenation of intermediates of the valine degradation pathway. Such a dual activity has not been observed with the animal IVD and may suggest a novel connection of the Leu and valine catabolism in plants.     

Regulation of [beta]-Methylcrotonyl-Coenzyme A Carboxylase Activity by Biotinylation of the Apoenzyme

Regulation of the expression of the gene(s) coding for the 78-kD, biotin-containing subunit of [beta]-methylcrotonyl-coenzyme A carboxylase (MCCase) was investigated in different organs of tomato (Lycopersicon esculantus) plants. The specific activity of MCCase is highest in extracts from roots, followed in descending order by ripe and ripening fruits, stems, and leaves. The specific activity is 10-fold higher in roots than in leaves. However, the steady-state levels of the 78-kD subunit of MCCase and its mRNA are approximately equal in both roots and leaves. Instead, the difference in MCCase activity between these two organs is directly correlated to the biotinylation status of the enzyme's biotin-containing subunit. Thus, the lower activity of MCCase in leaves is attributed to the reduced biotinylation of the biotin-containing subunit of the enzyme. Consistent with this model, a pool of nonbiotinylated enzyme is present in leaves, whereas the nonbiotinylated enzyme is undetectable in roots. The underbiotinylation of MCCase in leaves is not due to a lack of biotin in this organ, since the biotin concentration is 4- to 5-fold higher in leaves than in roots. These observations indicate that the posttranslational biotinylation of the biotin-containing sub-unit of MCCase is an important mechanism for regulating the organ-specific expression of MCCase activity.     

植物二酰甘油酰基转移酶基因(DGAT)研究进展

三酰甘油(TAG)是油料作物最主要的储藏脂类,二酰甘油酰基转移酶(DGAT,EC2.3.1.20)是TAG合成途径的限速酶,其主要作用是催化二酰甘油加上酰基脂肪酸形成三酰甘油.在植物中已发现了3种不同类型的DGAT基因,分别为DGAT1、DGAT2和DGAT3.该文对近年来国内外有关植物DGAT相关基因及其蛋白分类、定位、结构及其在脂肪酸合成、种子发育与萌发、幼苗发育、叶片新陈代谢等过程中的作用等研究进展进行综述.为提高油料作物种子油含量以及特定脂肪酸积累提供理论参考.     

Separation of the intermediates of leucine catabolism by high-performance liquid chromatography

A method has been developed for the separation of leucine, 2-ketoisocaproic acid, isovaleryl CoA, 3-methylcrotonyl CoA, 3-hydroxy-3-methylglutaryl CoA, 3-methylglutaconyl CoA, acetyl CoA, and acetoacetic acid by ion-exchange high-performance liquid chromatography. The analysis requires 180 min. Use of this method to assess the catabolism of radiolabeled leucine in normal cultured human skin fibroblasts shows that these cells do not accumulate CoA esters, but convert leucine mainly to 2-ketoisocaproic acid, glutamate, and hydroxyisovalerate. In the fibroblasts of a patient with maple syrup urine disease, only 2-ketoiscaproic acid is produced from leucine.     

2-Methylbutyryl CoA dehydrogenase from mitochondria of Ascarissuum and its relationship to NADH-dependent 2-methylcrotonyl CoA reduction

Acyl CoA dehydrogenase and electron-transfer flavoprotein have been isolated and partially purified from mitochondria of the anaerobic nematode, $\text{Ascaris}\text{suum}$. Dehydrogenase activity was greatest with 2-methylbutyryl CoA and the relative substrate specificities of the ascarid dehydrogenase(s) differ greatly from their mammalian counterparts. It appears that the ascarid dehydrogenase functions physiologically as a reductase, catalyzing the final step in the synthesis of branched-chain fatty acids. In fact, incubations of $\text{A}\text{.}\text{suum}$ mitochondrial membranes with electron-transfer flavoprotein, 2-methylbutyryl CoA dehydrogenase, 2-methylcrotonyl CoA and NADH resulted in a substantial, rotenone-sensitive, 2-methylbutyrate synthesis. These results suggest that the ascarid electron-transport chain and at least two soluble mitochondrial proteins are involved in the NADH-dependent reduction of 2-methylcrotonyl CoA.     

Co-expression of α and β subunits of the 3-methylcrotonyl-coenzyme A carboxylase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Pseudomonas aeruginosa is a versatile bacterium that can grow using citronellol or leucine as sole carbon source. For both compounds the degradation pathways converge at the key enzyme 3-methylcrotonyl coenzyme-A carboxylase (MCCase). This enzyme is a complex formed by two subunits (α and β), encoded by the liuD and liuB genes, respectively; both are essential for enzyme function. Previously, both subunits had been separately expressed and then the complex re-constituted, however this methodology is laborious and produces low yield of active enzyme. In this work, the MCCase subunits were co-expressed in the same plasmid and purified in one step by affinity chromatography using the LiuD-His tag protein, interacting with the LiuB-S tag recombinant protein. The purified enzyme lost most of the activity within few hours of storage. The co-expressed subunits formed an (αβ)₄ complex that suffered a modification of its oligomerization state after storage, which probably contributed to the loss on activity observed. The recombinant MCCase enzyme presented optimum pH and temperature values of 9.0 and 30o C, respectively. Functionally, MCCase showed Michaelian kinetics behavior with a K_m for its substrate and V_max of 168 μM and 430 nmoles mg⁻¹min⁻¹, respectively. The results suggest that the co-expression and co-purification of the subunits is a suitable procedure to obtain the active complex of the MCCase from Pseudomonas aeruginosa in a single step.     

A new insight into arable weed adaptive evolution: mutations endowing herbicide resistance also affect germination dynamics and seedling emergence

Background and Aims

Selective pressures exerted by agriculture on populations of arable weeds foster the evolution of adaptive traits. Germination and emergence dynamics and herbicide resistance are key adaptive traits. Herbicide resistance alleles can have pleiotropic effects on a weed''s life cycle. This study investigated the pleiotropic effects of three acetyl-coenzyme A carboxylase (ACCase) alleles endowing herbicide resistance on the seed-to-plant part of the life cycle of the grass weed Alopecurus myosuroides.

Methods

In each of two series of experiments, A. myosuroides populations with homogenized genetic backgrounds and segregating for Leu1781, Asn2041 or Gly2078 ACCase mutations which arose independently were used to compare germination dynamics, survival in the soil and seedling pre-emergence growth among seeds containing wild-type, heterozygous and homozygous mutant ACCase embryos.

Key Results

Asn2041 ACCase caused no significant effects. Gly2078 ACCase major effects were a co-dominant acceleration in seed germination (1·25- and 1·10-fold decrease in the time to reach 50 % germination (T₅₀) for homozygous and heterozygous mutant embryos, respectively). Segregation distortion against homozygous mutant embryos or a co-dominant increase in fatal germination was observed in one series of experiments. Leu1781 ACCase major effects were a co-dominant delay in seed germination (1·41- and 1·22-fold increase in T₅₀ for homozygous and heterozygous mutant embryos, respectively) associated with a substantial co-dominant decrease in fatal germination.

Conclusions

Under current agricultural systems, plants carrying Leu1781 or Gly2078 ACCase have a fitness advantage conferred by herbicide resistance that is enhanced or counterbalanced, respectively, by direct pleiotropic effects on the plant phenology. Pleiotropic effects associated with mutations endowing herbicide resistance undoubtedly play a significant role in the evolutionary dynamics of herbicide resistance in weed populations. Mutant ACCase alleles should also prove useful to investigate the role played by seed storage lipids in the control of seed dormancy and germination.     

A role for brassinosteroids in germination in Arabidopsis

This paper presents evidence that plant brassinosteroid (BR) hormones play a role in promoting germination. It has long been recognized that seed dormancy and germination are regulated by the plant hormones abscisic acid (ABA) and gibberellin (GA). These two hormones act antagonistically with each other. ABA induces seed dormancy in maturing embryos and inhibits germination of seeds. GA breaks seed dormancy and promotes germination. Severe mutations in GA biosynthetic genes in Arabidopsis, such as ga1-3, result in a requirement for GA application to germinate. Whereas previous work has shown that BRs play a critical role in controlling cell elongation, cell division, and skotomorphogenesis, no germination phenotypes have been reported in BR mutants. We show that BR rescues the germination phenotype of severe GA biosynthetic mutants and of the GA-insensitive mutant sleepy1. This result shows that BR stimulates germination and raises the possibility that BR is needed for normal germination. If true, we would expect to detect a germination phenotype in BR mutants. We found that BR mutants exhibit a germination phenotype in the presence of ABA. Germination of both the BR biosynthetic mutant det2-1 and the BR-insensitive mutant bri1-1 is more strongly inhibited by ABA than is germination of wild type. Thus, the BR signal is needed to overcome inhibition of germination by ABA. Taken together, these results point to a role for BRs in stimulating germination.