Diurnal rhythm of rat liver cytosolic 3-hydroxy-3-methylglutaryl-CoA synthase.
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ABSTRACT:
SUBMITTER: Royo T
PROVIDER: S-EPMC1130600 | biostudies-other | 1991 Nov
REPOSITORIES: biostudies-other
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Project description:The formation of carbon-carbon bonds via an acyl-enzyme intermediate plays a central role in fatty acid, polyketide, and isoprenoid biosynthesis. Uniquely among condensing enzymes, 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGS) catalyzes the formation of a carbon-carbon bond by activating the methyl group of an acetylated cysteine. This reaction is essential in Gram-positive bacteria, and represents the first committed step in human cholesterol biosynthesis. Reaction kinetics, isotope exchange, and mass spectroscopy suggest surprisingly that HMGS is able to catalyze the "backwards" reaction in solution, where HMG-CoA is cleaved to form acetoacetyl-CoA (AcAc-CoA) and acetate. Here, we trap a complex of acetylated HMGS from Staphylococcus aureus and bound acetoacetyl-CoA by cryo-cooling enzyme crystals at three different times during the course of its back-reaction with its physiological product (HMG-CoA). This nonphysiological "backwards" reaction is used to understand the details of the physiological reaction with regards to individual residues involved in catalysis and substrate/product binding. The structures suggest that an active-site glutamic acid (Glu-79) acts as a general base both in the condensation between acetoacetyl-CoA and the acetylated enzyme, and the hydrolytic release of HMG-CoA from the enzyme. The ability to trap this enzyme-intermediate complex may suggest a role for protein dynamics and the interplay between protomers during the normal course of catalysis.
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Project description:The mitochondrial sirtuin SIRT3 regulates metabolic homeostasis during fasting and calorie restriction. We identified mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase 2 (HMGCS2) as an acetylated protein and a possible target of SIRT3 in a proteomics survey in hepatic mitochondria from Sirt3(-/-) (SIRT3KO) mice. HMGCS2 is the rate-limiting step in ?-hydroxybutyrate synthesis and is hyperacetylated at lysines 310, 447, and 473 in the absence of SIRT3. HMGCS2 is deacetylated by SIRT3 in response to fasting in wild-type mice, but not in SIRT3KO mice. HMGCS2 is deacetylated in vitro when incubated with SIRT3 and in vivo by overexpression of SIRT3. Deacetylation of HMGCS2 lysines 310, 447, and 473 by incubation with wild-type SIRT3 or by mutation to arginine enhances its enzymatic activity. Molecular dynamics simulations show that in silico deacetylation of these three lysines causes conformational changes of HMGCS2 near the active site. Mice lacking SIRT3 show decreased ?-hydroxybutyrate levels during fasting. Our findings show SIRT3 regulates ketone body production during fasting and provide molecular insight into how protein acetylation can regulate enzymatic activity.
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Project description:In hypophysectomized and thyroidectomized rats, cytosolic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase activity, immunoreactive protein and mRNA levels were all considerably decreased. Administration of L-tri-iodothyronine (T3) resulted in a large increase in all three in hypophysectomized rats and in only a 2-fold increase (reaching the values of control rats) in thyroidectomized rats.
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Project description:Tripterygium wilfordii is a traditional Chinese medical plant used to treat rheumatoid arthritis and cancer. The main bioactive compounds of the plant are diterpenoids and triterpenoids. 3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) catalyses the reaction of acetoacetyl-CoA to 3-hydroxy-3-methylglutaryl-CoA, which is the first committed enzyme in the mevalonate (MVA) pathway. The sequence information of HMGS in Tripterygium wilfordii is a basic resource necessary for studying the terpenoids in the plant. In this paper, full-length cDNA encoding HMGS was isolated from Tripterygium wilfordii (abbreviated TwHMGS, GenBank accession number: KM978213). The full length of TwHMGS is 1814 bp, and the gene encodes a protein with 465 amino acids. Sequence comparison revealed that TwHMGS exhibits high similarity to HMGSs of other plants. The tissue expression patterns revealed that the expression level of TwHMGS is highest in the stems and lowest in the roots. Induced expression of TwHMGS can be induced by MeJA, and the expression level is highest 4 h after induction. The functional complement assays in the YML126C knockout yeast demonstrated that TwHMGS participates in yeast terpenoid biosynthesis.
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Project description:3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) is the second enzyme in the mevalonate pathway. While the recombinant Brassica juncea HMGS1 (BjHMGS1) mutant S359A displayed 10-fold higher enzyme activity than wild-type (wt) BjHMGS1, transgenic tobacco overexpressing S359A (OE-S359A) exhibited greater sterol content, growth rate and seed yield than OE-wtBjHMGS1. To explore the mechanism of HMGS and its mutant (S359A) in promotion of plant growth, SWATH-MS quantitative proteomics analysis was performed.
2021-01-04 | PXD006869 | Pride
Project description:'Initial' and 'total' activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) were measured in cold-clamped samples of liver from rats at 2h intervals throughout the 24h light/dark cycle. Initial activities were obtained in microsomes (microsomal fractions) isolated and assayed in the presence of 100mM-KF, whereas 'total' activities were measured in microsomes prepared from the same homogenates but washed free of KF and incubated with exogenous partially purified rat liver protein phosphatase. The initial/total-activity ratio for HMG-CoA reductase underwent a diurnal cycle, which had a nadir 4h into the light phase (when initial activity was 28% of total activity) and a peak 12h later, i.e. 4h into the dark phase (when initial activity was 80% of total activity). These low and high points of the cycle were separated by gradual steady changes in the ratio. The characteristics of this diurnal cycle were different from those of the cycle observed for total activity, which had a plateau of high activity between 2 and 10h into the dark cycle preceded and succeeded by a very rapid increase and decrease, respectively, in the total activity of HMG-CoA reductase. The combination of the two cycles resulted in the dampening of the resultant cycle for the initial or effective activity of HMG-CoA reductase, such that the changes in initial activity around the beginning and and end of the dark phase were more gradual than would otherwise have been the case if the initial/total-activity ratio for HMG-CoA reductase were constant throughout the diurnal cycle. The physiological implications of the observed diurnal variation in the fraction of hepatic HMG-CoA reductase in the active form are discussed.
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Project description:The tissue-specific expression of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase gene was studied in 15-day-old suckling rats. The mRNA and protein were present in liver, intestine and kidney, but were absent from brain, heart, skeletal muscles, brown and white adipose tissues. Kidney-cortex mitochondria from suckling rats were able to produce low amounts of ketone bodies from oleate. Hepatic, intestinal and renal HMG-CoA synthase mRNA levels increased slowly during foetal life and markedly after birth. The postnatal increase in liver HMG-CoA synthase mRNA could be due to the increase in plasma glucagon levels, since it rapidly induced the accumulation of HMG-CoA synthase mRNA in cultured foetal hepatocytes. Hepatic, intestinal and renal HMG-CoA synthase mRNA levels remained elevated throughout the suckling period or in rats weaned on to a high-fat carbohydrate-free diet (HF), but decreased by 50% in the liver and totally disappeared from the intestine and the kidney of rats weaned on to a high-carbohydrate low-fat diet (HC). When HC-weaned rats were fed on a HF-diet for a week, HMG-CoA synthase mRNA was re-induced in the intestine and the kidney. The role of hormones and nutrients in the regulation of HMG-CoA synthase gene expression is discussed.
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Project description:Sandalwood (Santalum album L.) is famous for its unique fragrance derived from the essential oil of heartwood, whose major components are santalols. To understand the mechanism underlying the biosynthesis of santalols, in this study, we cloned two related genes involved in the mevalonate pathway in S. album coding for acetyl-CoA C-acetyl transferase (AACT) and 3-hydroxy-3-methyglutary-CoA synthase (HMGS). These genes were characterized and functionally analyzed, and their expression profiles were also assessed. An AACT gene designated as SaAACT (GenBank accession No. MH018694) and a HMGS gene designated as SaHMGS (GenBank accession No. MH018695) were successfully cloned from S. album. The deduced SaAACT and SaHMGS proteins contain 415 and 470 amino acids, and the corresponding size of their open-reading frames is 1538 bp and 1807 bp, respectively. Phylogenetic trees showed that the SaAACT protein had the closest relationship with AACT from Hevea brasiliensis and the SaHMGS proteins had the highest homology with HMGS from Siraitia grosvenorii. Functional complementation of SaAACT and SaHMGS in a mutant yeast strain deficient in these proteins confirmed that SaAACT and SaHMGS cDNA encodes functional SaAACT and SaHMGS that mediate mevalonate biosynthesis in yeast. Tissue-specific expression analysis revealed that both genes were constitutively expressed in all examined tissues (roots, sapwood, heartwood, young leaves, mature leaves and shoots) of S. album, both genes showing highest expression in roots. After S. album seedlings were treated with 100 μM methyl jasmonate, the expression levels of SaAACT and SaHMGS genes increased, suggesting that these genes were responsive to this elicitor. These studies provide insight that would allow further analysis of the role of genes related to the sandalwood mevalonate pathway in the regulation of biosynthesis of sandalwood terpenoids and a deeper understanding of the molecular mechanism of santalol biosynthesis.
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Project description:Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency (mHS deficiency) is a rare autosomal recessive inborn error of ketogenesis caused by a mutation in the HMGCS2 gene, which is characterized by non-(hypo)-ketotic hypoglycemia, lethargy, and hepatomegaly during acute infection and/or prolonged fasting. Clinical presentations are similar to fatty acid oxidation defects; however, diagnosis of mHS deficiency is difficult because of poor biochemical markers. We report the case of a 12-month-old Japanese boy with mHS deficiency who presented with a coma, and hepatomegaly, but no hypoglycemia after a febrile episode and poor oral intake. Metabolic acidosis and severe fatty liver were observed. Serum acylcarnitine analysis revealed a slightly decreased free carnitine (C0) level and an increased acetylcarnitine (C2) level. Urinary organic acid analysis revealed hypoketotic dicarboxylic aciduria, and increased excretions of glutarate, and, retrospectively, 4-hydroxy-6-methyl-2-pyrone. Although the patient did not present with hypoglycemia, the severe fatty liver and elevated free fatty acids to total ketone bodies ratio strongly suggested an inborn error of ketogenesis. In the analysis of the HMGCS2 gene, compound heterozygous mutations of c.130_131ins C (L44PfsX29) and c.1156_1157insC (L386PfsX73) were identified, which led to the diagnosis of mHS deficiency. He had recovered without any complication by the therapy, including intravenous glucose infusion. Unlike the previously reported cases of mHS deficiency, our case did not present with hypoglycemia and the fatty liver lasted over several months. mHS deficiency should be taken into consideration when a patient has severe metabolic acidosis and fatty liver with no or subtle ketosis, even without hypoglycemia.
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Project description:The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase catalyzes the conversion of HMG-CoA to mevalonate, a four-electron oxidoreduction that is the rate-limiting step in the synthesis of cholesterol and other isoprenoids. The enzyme is found in eukaryotes and prokaryotes; and phylogenetic analysis has revealed two classes of HMG-CoA reductase, the Class I enzymes of eukaryotes and some archaea and the Class II enzymes of eubacteria and certain other archaea. Three-dimensional structures of the catalytic domain of HMG-CoA reductases from humans and from the bacterium Pseudomonas mevalonii, in conjunction with site-directed mutagenesis studies, have revealed details of the mechanism of catalysis. The reaction catalyzed by human HMG-CoA reductase is a target for anti-hypercholesterolemic drugs (statins), which are intended to lower cholesterol levels in serum. Eukaryotic forms of the enzyme are anchored to the endoplasmic reticulum, whereas the prokaryotic enzymes are soluble. Probably because of its critical role in cellular cholesterol homeostasis, mammalian HMG-CoA reductase is extensively regulated at the transcriptional, translational, and post-translational levels.
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