A visible wavelength spectrophotometric assay suitable for high-throughput screening of 3-hydroxy-3-methylglutaryl-CoA synthase

Skaff, D. Andrew; Miziorko, Henry M.

doi:10.1016/j.ab.2009.08.030

Cited by 22 publications

(17 citation statements)

References 19 publications

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“…Steady state kinetic analysis was carried out using acetyl-CoA and acetoacetyl-CoA as substrates and activity was measured by release of CoA-SH at increasing concentrations of acetyl-CoA (Figure 7D). While Km and Vmax for WT HMGCS2 kinetic activity was reasonably similar to previous reports (Andrew Skaff and Miziorko, 2010; Shimazu et al, 2010) the individual mutants (K83E, K310E) and the double mutant (K83/310E) exhibited a complete loss of enzymatic activity (Figure 7D). Given the robust changes in succinylation at K83 and K310 in Sirt5 −/− mice, their close proximity to the substrate binding pocket, and the loss of function when replaced with a negatively charged amino acid, our data support the model that lysine succinylation and desuccinylation by SIRT5 can regulate HMGCS2 activity through disrupting and restoring the binding pocket for phosphate groups of acetyl-CoA respectively.…”

Section: Resultssupporting

confidence: 90%

SIRT5 Regulates the Mitochondrial Lysine Succinylome and Metabolic Networks

et al. 2013

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Summary Reversible posttranslational modifications are emerging as critical regulators of mitochondrial proteins and metabolism. Here, we use a label-free quantitative proteomic approach to characterize the lysine succinylome in liver mitochondria and its regulation by the desuccinylase SIRT5. A total of 1190 unique sites were identified as succinylated, and 386 sites across 140 proteins representing several metabolic pathways including β-oxidation and ketogenesis were significantly hypersuccinylated in Sirt5−/− animals. Loss of SIRT5 leads to accumulation of medium- and long-chain acylcarnitines and decreased β-hydroxybutyrate production in vivo. In addition, we demonstrate that SIRT5 regulates succinylation of the rate-limiting ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) both in vivo and in vitro. Finally, mutation of hypersuccinylated residues K83 and K310 on HMGCS2 to glutamic acid strongly inhibits enzymatic activity. Taken together, these findings establish SIRT5 as a global regulator of lysine succinylation in mitochondria and present a mechanism for inhibition of ketogenesis through HMGCS2.

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Section: Resultssupporting

confidence: 90%

SIRT5 Regulates the Mitochondrial Lysine Succinylome and Metabolic Networks

et al. 2013

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“…65. Acetylation reaction, monitored at 412 nm and performed at 30 °C, was started by the addition of 50 μl of the NatA complex (200 nM, Naa15-Naa10ΔC-His 6 ) to 50 μl of pre-incubated reaction solution containing 50 mM HEPES pH 7.4, 2 mM EDTA, 1 mM 5,5′-dithio-bis-2-nitrobenzoic acid, 1.5 mM SESS-peptide (PSL Peptide Specialty Laboratories GmbH, Heidelberg) and various concentrations of acetyl-CoA.…”

Section: Methodsmentioning

confidence: 99%

Structural basis of HypK regulating N-terminal acetylation by the NatA complex

et al. 2017

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In eukaryotes, N-terminal acetylation is one of the most common protein modifications involved in a wide range of biological processes. Most N-acetyltransferase complexes (NATs) act co-translationally, with the heterodimeric NatA complex modifying the majority of substrate proteins. Here we show that the Huntingtin yeast two-hybrid protein K (HypK) binds tightly to the NatA complex comprising the auxiliary subunit Naa15 and the catalytic subunit Naa10. The crystal structures of NatA bound to HypK or to a N-terminal deletion variant of HypK were determined without or with a bi-substrate analogue, respectively. The HypK C-terminal region is responsible for high-affinity interaction with the C-terminal part of Naa15. In combination with acetylation assays, the HypK N-terminal region is identified as a negative regulator of the NatA acetylation activity. Our study provides mechanistic insights into the regulation of this pivotal protein modification.

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“…HMGCS2 enzymatic activity was measured by monitoring the conversion of acetyl-CoA and acetoacetyl-CoA to 3-hydroxy-3-methylglutaryl-CoA, as measured by DTNB detection of CoASH (Andrew Skaff and Miziorko, 2010). Assay mixtures contained 100 mM Tris-Cl (pH 8.0), 130 μM DTNB (A 412 nm = 13.6 mM −1 ), 10–1400 μM acetyl-CoA, 10 μM acetoacetyl-CoA, and 1 μg of enzyme in a total volume of 200 μL.…”

Section: Methodsmentioning

confidence: 99%

SIRT3 Deacetylates Mitochondrial 3-Hydroxy-3-Methylglutaryl CoA Synthase 2 and Regulates Ketone Body Production

et al. 2010

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SUMMARY 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 β-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 β-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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A visible wavelength spectrophotometric assay suitable for high-throughput screening of 3-hydroxy-3-methylglutaryl-CoA synthase

Cited by 22 publications

References 19 publications

SIRT5 Regulates the Mitochondrial Lysine Succinylome and Metabolic Networks

SIRT5 Regulates the Mitochondrial Lysine Succinylome and Metabolic Networks

Structural basis of HypK regulating N-terminal acetylation by the NatA complex

SIRT3 Deacetylates Mitochondrial 3-Hydroxy-3-Methylglutaryl CoA Synthase 2 and Regulates Ketone Body Production

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