Mevalonate Shunt Research Articles

Genetic dissection of methylcrotonyl CoA carboxylase indicates a complex role for mitochondrial leucine catabolism during seed development and germination

3-methylcrotonyl CoA carboxylase (MCCase) is a nuclear-encoded, mitochondrial-localized biotin-containing enzyme. The reaction catalyzed by this enzyme is required for leucine (Leu) catabolism, and it may also play a role in the catabolism of isoprenoids and the mevalonate shunt. In Arabidopsis, two MCCase subunits (the biotinylated MCCA subunit and the non-biotinylated MCCB subunit) are each encoded by single genes (At1g03090 and At4g34030, respectively). A reverse genetic approach was used to assess the physiological role of MCCase in plants. We recovered and characterized T-DNA and transposon-tagged knockout alleles of the MCCA and MCCB genes. Metabolite profiling studies indicate that mutations in either MCCA or MCCB block mitochondrial Leu catabolism, as inferred from the increased accumulation of Leu. Under light deprivation conditions, the hyper-accumulation of Leu, 3-methylcrotonyl CoA and isovaleryl CoA indicates that mitochondrial and peroxisomal Leu catabolism pathways are independently regulated. This biochemical block in mitochondrial Leu catabolism is associated with an impaired reproductive growth phenotype, which includes aberrant flower and silique development and decreased seed germination. The decreased seed germination phenotype is only observed for homozygous mutant seeds collected from a parent plant that is itself homozygous, but not from a parent plant that is heterozygous. These characterizations may shed light on the role of catabolic processes in growth and development, an area of plant biology that is poorly understood.

Fungal Metabolic Model for 3-Methylcrotonyl-CoA Carboxylase Deficiency

Aspergillus nidulans is able to use Leu as the sole carbon source through a metabolic pathway leading to acetyl-CoA and acetoacetate that is homologous to that used by humans. mccA and mccB, the genes encoding the subunits of 3-methylcrotonyl-CoA carboxylase, are clustered with ivdA encoding isovaleryl-CoA dehydrogenase, a third gene of the Leu catabolic pathway, on the left arm of chromosome III. Their transcription is induced by Leu and other hydrophobic amino acids and repressed by glucose. Phenotypically indistinguishable DeltamccA, DeltamccB, and DeltamccA DeltamccB mutations prevent growth on Leu but not on lactose or other amino acids, formally demonstrating in vivo the specific involvement of 3-methylcrotonyl-CoA carboxylase in Leu catabolism. Growth of mcc mutants on lactose plus Leu is impaired, indicating that Leu metabolite(s) accumulation resulting from the metabolic block is toxic. Human patients carrying loss-of-function mutations in the genes encoding the subunits of 3-methylcrotonyl-CoA carboxylase suffer from methylcrotonylglycinuria. Gas chromatography/mass spectrometry analysis of culture supernatants revealed that fungal Deltamcc strains accumulate 3-hydroxyisovaleric acid, one of the diagnostic compounds in the urine of these patients, illustrating the remarkably similar consequences of equivalent genetic errors of metabolism in fungi and humans. We use our fungal model(s) for methylcrotonylglycinuria to show accumulation of 3-hydroxyisovalerate on transfer of 3-methylcrotonyl-CoA carboxylase-deficient strains to the isoprenoid precursors acetate, 3-hydroxy-3-methylglutarate, or mevalonate. This represents the first reported genetic evidence for the existence of a metabolic link involving 3-methylcrotonyl-CoA carboxylase between isoprenoid biosynthesis and Leu catabolism, providing additional support to the mevalonate shunt proposed previously (Edmond, J., and Popják, G. (1974) J. Biol. Chem. 249, 66-71).

Metabolic and environmental regulation of 3-methylcrotonyl-coenzyme A carboxylase expression in Arabidopsis.

3-Methylcrotonyl-coenzyme A carboxylase (MCCase) is a nuclear-encoded, mitochondrial biotin-containing enzyme composed of two types of subunits: the biotinylated MCC-A subunit (encoded by the gene At1g03090) and the non-biotinylated MCC-B subunit (encoded by the gene At4g34030). The major metabolic role of MCCase is in the mitochondrial catabolism of leucine, and it also might function in the catabolism of isoprenoids and the mevalonate shunt. In the work presented herein, the single-copy gene encoding the Arabidopsis MCC-A subunit was isolated and characterized. It contains 15 exons separated by 14 introns. We examined the expression of the single-copy MCC-A and MCC-B genes in Arabidopsis by monitoring the accumulation of the two protein and mRNA products. In addition, the expression of these two genes was studied in transgenic plants containing the 1.1- and 1.0-kb 5' upstream sequences of the two MCCase subunit genes, respectively, fused to the beta-glucuronidase gene. Light deprivation induces MCCase expression, which is suppressed by exogenous carbohydrates, especially sucrose. Several lines of evidence indicate that the suppressor of MCCase expression is synthesized in illuminated photosynthetic organs, and can be translocated to other organs to regulate MCCase expression. These results are consistent with the hypothesis that the suppressor of MCCase expression is a carbohydrate, perhaps sucrose or a carbohydrate metabolite. We conclude that MCCase expression is primarily controlled at the level of gene transcription and regulated by a complex interplay between environmental and metabolic signals. The observed expression patterns may indicate that one of the physiological roles of MCCase is to maintain the carbon status of the organism, possibly via the catabolism of leucine.

What is the origin of 3-methylglutaconic acid?

Increased urinary excretion of 3-methylglutaconic acid (3-MGC) and to a lesser extent 3-methylglutaric acid (3-MGR) is a not uncommon finding in children who are being investigated for presumed inborn errors of metabolism. During the last 5 years we have identified 70 such individuals. None of these had the characteristic clinical and laboratory findings of the three well-defined syndromes of 3-methylglutaconic aciduria (types I, II and III); all therefore were classified as type IV, the 'unspecified form' of Sweetman and Williams (1995). The origin of 3-MGC and its reduced metabolite 3-MGR in type IV methylglutaconic aciduria is not known (nor indeed is it for types II and III). The only proven enzymatic defect is in type I, the rarest of the four types, where 3-methylglutaconyl-CoA hydratase, an enzyme of the leucine catabolic pathway, is deficient. It has been suggested that 3-MGC (and thus 3-MGR) could occur as by-products of steroid synthesis (Kelley et al 1991) via the so-called mevalonate shunt pathway. Although the results of initial investigations of steroid biosynthesis in methylglutaconic aciduria were not promising (Ibel et al 1993), support was lent to this hypothesis by our finding that increased excretion of 3-MGC and 3-MGR was a normal finding during pregnancy when an increased flux through steroid biosynthesis would be expected (Walsh et al 1997). To further test this hypothesis, we examined urine specimens from patients with congenital adrenal hyperplasia (CAH), who would also be expected to have increased flux through the steroid biosynthetic pathways when under suboptimal control.

3-methylglutaconic acidemia in Smith-Lemli-Opitz syndrome.

The branched-chain organic acid, 3-methylglutaconic acid, is an intermediate (as the CoA thioester) in the leucine degradative pathway as well as the mevalonate shunt, a pathway that links isoprenoid metabolism with mitochondrial acetyl-CoA metabolism. Because the majority of patients with abnormal 3-methylglutaconic aciduria or acidemia appear to have normal leucine metabolism, we have speculated that some patients with 3-methylglutaconic aciduria may have defects of polyisoprenoid or sterol biosynthesis leading to overflow of isoprenoid precursors to 3-methylglutaconate via the mevalonate shunt. We therefore measured plasma levels of 3-methylglutaconic acid in patients with a known defect of sterol biosynthesis, Smith-Lemli-Opitz syndrome, and found that the patients with the lowest cholesterol levels had abnormally increased plasma levels of 3-methylglutaconic acid, similar in magnitude to those of patients with idiopathic 3-methylglutaconic aciduria. This finding suggests that some patients with unexplained 3-methylglutaconic aciduria may have defects of isoprenoid or sterol biosynthesis underlying their abnormal organic aciduria.

The effects of excess exogenous mevalonic acid on sterol and steryl ester biosynthesis in celery (Apium graveolens) cell suspension cultures

Cell suspension cultures of celery ( Apium graveolens) have been used to investigate the fate of excess added mevalonic acid and the effects on the amounts of free sterols and steryl esters in the cells. Radiolabelling studies using [2- 14C] MVA showed that the main effect of excess MVA (5 mM) was an increase in the synthesis of steryl esters. Further analysis revealed that esterified 4,4-dimethylsterols were more highly labelled than 4-mono- and 4-desmethylsterols in MVA-treated cells, suggesting a restriction in the flow of carbon downstream from cycloartenol and 24-methylenecycloartanol, and thus indicating the presence of a post-mevalonic acid control point in the sterol biosynthesis pathway. The total fatty acids recovered from the celery cell lipids showed a very low incorporation of radioactivity from the [2- 14C] mevalonate, indicating that a limited breakdown of excess mevalonate to acetate may occur in these cells, possibly by the operation of the mevalonate shunt. However, this does not appear to be a major route for the disposal of excess mevalonate. Analysis of the free sterols and steryl esters showed that the administration of excess exogenous MVA (5 mM) to Apium graveolens cells caused a two-fold increase in the steryl ester content when compared with control cells, although there was little effect on the free sterol content or composition. When excess MVA was administered in conjunction with the sterol biosynthesis inhibitor paclobutrazol (50 μM), the steryl ester content increased almost three-fold. It is possible that, as well as providing a means of storage of excess sterols, the esterification of sterols may have a regulatory role in sterol biosynthesis, by controlling the rate of C-4 demethylation of precursor 4,4-dimethylsterols.

T-2 toxin biosynthesis: origin of the isovalerate side chain

In biosynthetic incorporation experiments 13 C-labeled leucine was shown to be incorporated into the C-8 isovalerates side chain of T-2 toxin. Other incorporation experiments and implications to the mevalonate shunt pathway as well as to directed biosynthetic studies are also discussed

Role of the kidneys in the metabolism of circulating mevalonate in humans.

Previous studies in several animal species have demonstrated that the kidneys are the primary site of mevalonate metabolism by the oxidative or shunt pathway. To determine the role of the human kidney in mevalonate oxidation, we studied mevalonate shunt activity in patients undergoing hemodialysis for varying degrees of renal failure. Surprisingly, at least half of the uremic patients and even anephric patients had normal ability to oxidize mevalonate by the shunt pathway. In addition, we found a strong negative correlation (R = -0.94) between mevalonate shunt activity and serum phosphorus levels in uremic patients. The resulting inhibition of mevalonate oxidation by high serum phosphorus levels was reversed by lowering the serum phosphorus in one patient. Finally, a positive correlation was found between mevalonate oxidation and serum PTH levels. The results of this study suggest that, in humans, extrarenal tissues can be major contributors to mevalonate oxidation. It is therefore probable that in humans, in contrast to other animals, the kidney is not the primary site of mevalonate metabolism by this oxidative pathway. Finally, the strong negative correlation between serum phosphorus levels and the ability of uremic patients to oxidize mevalonate suggests a regulatory role for the phosphate ion in the mevalonate shunt pathway.

Evidence for an underestimation of the shunt pathway of mevalonate metabolism in slices of livers and kidneys from fasted rats and rats in diabetic ketosis

Yields of (14)CO(2) from [2-(14)C]mevalonate and [5-(14)C]mevalonate have been used by others to estimate the activity of the non-sterol-forming pathway, also called the mevalonate shunt pathway, and yields of (14)C in sterols have been used to estimate the activity of the sterol-forming pathway. Both these pathways operate following the conversion of carbon 1 of mevalonate to CO(2). The estimations of the shunt pathway contribution are dependent upon the fractions of carbons 2 and 5 of mevalonate that are oxidized to CO(2) in the Krebs cycle after leaving the pathway. Unless all of carbons 2 and 5 are oxidized to CO(2), the estimates are minimal. The metabolism of mevalonate has now been examined in slices of livers and kidneys from fasted rats and rats in diabetic ketosis. Yields of (14)CO(2) from [1-(14)C]mevalonate are used as the measure of the contributions of all the pathways by which carbon 1 of mevalonate is converted to CO(2). Yields of (3)H-labeled nonsaponifiable lipids from [5-(3)H]mevalonate are used as the measure of the sterol-forming pathway. The differences in these yields are then taken as the measure of the non-sterol-forming pathway or pathways. Yields of (14)CO(2) from [1-(14)C]mevalonate markedly exceeded the sum of the yields of (14)C in CO(2) and nonsaponifiable lipids from either [2-(14)C]mevalonate or [5-(14)C]mevalonate. Therefore, in liver and kidney, under the conditions of this study, either one or more pathways other than the shunt pathway, by which mevalonate can be metabolized to other than sterols, is operative to a marked degree, or estimates of the shunt pathway's contributions as judged by yields of (14)CO(2) from [2-(14)C]mevalonate and [5-(14)C]mevalonate are significantly underestimated.-Brady, P. S., W. C. Schumann, S. Ohgaku, R. F. Scofield, and B. R. Landau. Evidence for an underestimation of the shunt pathway of mevalonate metabolism in slices of livers and kidneys from fasted rats and rats in diabetic ketosis.

Impaired renal mevalonate metabolism in nephrotic syndrome: A stimulus for increased hepatic cholesterogenesis independent of GFR and hypoalbuminemia

Increased hepatic lipogenesis in the nephrotic syndrome is not adequately explained by hypoalbuminemia. In this disorder an enhanced delivery of the cholesterol precursor mevalonic acid (MVA) to the liver may be an unidentified stimulus to cholesterogenesis. Since the kidneys are the major site of mevalonate excretion and metabolism by either the sterol or nonsterol shunt pathways, an impairment of any of these metabolic alternatives could result in redistribution of mevalonate to the liver. Male Sprague-Dawley rats rendered nephrotic by puromycin aminonucleoside had their kidneys perfused with Krebs-Henseleit-bicarbonate buffer containing albumin, glucose and 5- 14C-MVA. The number five carbon label was utilized so that any 14CO 2 produced would represent mevalonate shunt pathway activity. The isolated perfused kidney was used to eliminate confounding variables. In eight control kidneys perfused for 2 hr 62 ± 2% of the MVA was removed from the perfusate compared to 50 ± 2% in five nephrotic kidneys ( p < .006). Urinary MVA recovery was 22 ± 2% in controls, 15 ± 1% in nephrotics ( p < .05). The incorportion of 14C into renal tissue lipids was not different in the two groups. Recovery of 14CO 2 was two times greater in controls than in nephrotics ( p < .006). Inulin clearance per gram of kidney and sodium reabsorption were similar for the two groups. Isolated perfused kidneys from nephrotic rats metabolize MVA abnormally such that less is excreted, less is oxidized, and more is available for recirculation to the liver. This occurs independently of hypoalbuminemia, a change in glomerular filtration rate, or an overt histo-pathologic lesion. These events create an environment for increased hepatic cholesterol synthesis.