3-Methylglutaconyl-CoA

Serine dehydratase Enoyl-CoA hydratase Methylglutaconyl-CoA hydratase Cystathionine p-synthase [PLP]... 

Substrates and products are separated by reversed-phase chromatography at 45°C on a Nucleosil Qs column (4.6 mm X 250 mm). For assay of acetyl-coenzyme A carboxylase, propionyl-coenzyme A carboxylase, and 3-methylcrotonyl-coenzyme A carboxylase, a linear gradient from solvent A (0.1 M sodium phosphate buffer, pH 2.1) to solvent B (methanol-solvent A, 80 20, v/v) was applied in 15 minutes at a flow rate of 1.5 mL/min. Quantitation was based on the absorbance of the product (malonyl-CoA, methylmalonyl-CoA, and 3-methylglutaconyl-CoA, respectively) at 260 nm. For assay of pyruvate carboxylase, pyruvate was separated by isocratic elution using 0.1 M sodium phosphate buffer (pH 2.1) containing 0.1 M sodium sulfate. Quantitation was based on the disappearance of pyruvate as followed at 210 nm. 

Methylcrotonyl CoA Carboxylase Methylcrotonyl CoA carboxylase catalyzes the conversion of methylcrotonyl CoA, arising from the catabolism of leucine, to methylglutaconyl CoA. This in turn undergoes hydroxy-lation catalyzed by crotonase, yielding hydroxymethyl-glutaryl CoA, which is cleaved to acetyl CoA and acetoacetate. 

The isovaleryl CoA derived from leucine is dehydrogenated to yield -methylcrotonyl CoA. This oxidation is catalyzed by isovaleryl CoA dehydrogenase. The hydrogen acceptor is FAD, as in the analogous reaction in fatty acid oxidation that is catalyzed by acyl CoA dehydrogenase. -Methylglutaconyl CoA is then formed by the carboxylation of P-methylcrotonyl CoA at the expense of the hydrolysis of a molecule of ATP. As might be expected, the carboxylation mechanism of P-methylcrotonyl CoA carboxylase is similar to that of pyruvate carboxylase and acetyl CoA carboxylase. 

P-Methylglutaconyl CoA is then hydrated to form 3-hydroxy-3-methylglutaryl CoA, which is cleaved into acetyl CoA and acetoacetate. This reaction has already been discussed in regard to the formation of ketone bodies from fatty acids (Section 22.3.5). 

Methylglutaryl-CoA hydratase (EC 4.2.1.18) catalyzes the reversible dehydration of HMG-CoA into 3-methylglutaconyl-CoA and is known from mammalian cells to be involved in leucine metabolism. This enzyme was detected in C. roseus suspension cultured cells and partially purified (87). Further enzymes channeling HMG-CoA away from isoprenoid biosynthesis in C. roseus cells were detected by a selective HPLC system showing that the CoA-esters may be rapidly dephosphorylated on the 3 -position by the action of 3 -nucleotidases (87). 

Methylglutaconyl-CoA (MG-CoA) hydratase (EC 4.2.1.18) activity yields MG-CoA from the substrate HMG-CoA. As for HMG-CoA lyase, this enzyme may compete with terpenoid biosynthesis. In mammalian cells this enzyme is involved in leucine metabolism. The activity of the enzyme was detected in various plant cell cultures, among others Arabidopsis thaliana. The C. roseus enzyme was partially purified by ammonium sulfate precipitation, ion- exchange and hydrcxyapatite chromatography, and gel filtration [5]. It is relatively stable and does not require cofactors. 

The carboxylation of methylcrotonyl CoA to form methylglutaconyl-CoA is catalyzed by MCCase. The role of this reaction in plant metabolism is as yet unclear. However, extrapolation from studies with animals and bacteria would suggest that MCCase may have a role in a set of metabolic processes that link amino acid and lipid metabolism. Namely, MCCase may be involved in the mitochondrial metabolism of leucine, mevalonate and noncyclic isoprenoids. 

The inborn errors of L-leucine catabolism present biochemically with branched-chain amino and/or organic aciduria [1]. These disorders include maple syrup disease (MSD branched-chain a-ketoacid dehydrogenase (BCKD) deficiency), isovaleric acidemia (isovaleryl-coenzyme A (CoA) dehydrogenase deficiency), isolated 3-methylcrotonyl-CoA carboxylase deficiency, the 3-methylglutaconic acidurias (3-methylglutaconyl-CoA hydratase deficiency, Barth syndrome, and other disorders in which the primary defect has not been demonstrated), and 3-hydroxy-3-methylglutaric aciduria (3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase deficiency). 

Methylglutaconk aciduria type I (3"methylglutaconyl-CoA hydratase deficiency) WBC, FB 250950... 

Fig. 6.1. The L-leucine degradative pathway. Reactions for which inherited metabolic disorders have not been conclusively identified include A, leucine-isoleucine aminotransferase and the majority of the 3-methylglutaconic acidurias (6.6-6.7). 6.1, Branched-chain a-ketoacid dehydrogenase (BCKD) complex, a reaction also occurring in the initial steps of L-isoleucine and L-valine degradation 6.2, isovaleryl-CoA dehydrogenase 6.3, 3-methylcrotonyl-CoA carboxylase 6.4, 3-methylglutaconyl-CoA hydra-tase 6.8, HMG-CoA lyase. Pathologic urinary metabolites used as specific markers in the differential diagnosis are presented in squares. Abbreviation Co A, coenzyme A...

Table 6.5. Barth syndrome (Xdinked 3-methylglutaconk aciduria, normal 3-methylglutaconyl-CoA hydratase activity) ...

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