ABSTRACT
SIRT1 and SIRT3 are NAD+-dependent protein deacetylases that are evolutionarily conserved across mammals. These proteins are located in the cytoplasm/nucleus and mitochondria, respectively. Previous reports demonstrated that human SIRT1 deacetylates Acetyl-CoA Synthase 1 (AceCS1) in the cytoplasm, whereas SIRT3 deacetylates the homologous Acetyl-CoA Synthase 2 (AceCS2) in the mitochondria. We recently showed that 3-hydroxy-3-methylglutaryl CoA synthase 2 (HMGCS2) is deacetylated by SIRT3 in mitochondria, and we demonstrate here that SIRT1 deacetylates the homologous 3-hydroxy-3-methylglutaryl CoA synthase 1 (HMGCS1) in the cytoplasm. This novel pattern of substrate homology between cytoplasmic SIRT1 and mitochondrial SIRT3 suggests that considering evolutionary relationships between the sirtuins and their substrates may help to identify and understand the functions and interactions of this gene family. In this perspective, we take a first step by characterizing the evolutionary history of the sirtuins and these substrate families.
Subject(s)
Acetate-CoA Ligase/metabolism , Evolution, Molecular , Hydroxymethylglutaryl-CoA Synthase/metabolism , Isoenzymes/metabolism , Sirtuin 1/metabolism , Sirtuin 3/metabolism , Acetate-CoA Ligase/classification , Acetate-CoA Ligase/genetics , Acetylation , Animals , Humans , Hydroxymethylglutaryl-CoA Synthase/classification , Hydroxymethylglutaryl-CoA Synthase/genetics , Isoenzymes/classification , Isoenzymes/genetics , Phylogeny , Sirtuin 1/classification , Sirtuin 1/genetics , Sirtuin 3/classification , Sirtuin 3/geneticsABSTRACT
In vitro analysis of natural product biosynthetic gene products isolated from unculturable symbiotic bacteria is necessary to probe the functionalities of these enzymes. Herein, we report the biochemical characterization of BryR, the 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGS) homolog implicated in ß-branching at C13 and C21 of the core ring system from the bryostatin metabolic pathway (Bry). We confirmed the activity of BryR using two complementary methods, radio-SDS PAGE, and Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS). The activity of BryR depended on pairing of the native acetoacetyl-BryM3 acceptor acyl carrier protein (ACP) with an appropriate donor acetyl-ACP from a heterologous HMGS cassette. Additionally, the ability of BryR to discriminate between various ACPs was assessed using a surface plasmon resonance (SPR)-based protein-protein binding assay. Our data suggest that specificity for a protein-bound acyl group is a distinguishing feature between HMGS homologs found in PKS or PKS/NRPS biosynthetic pathways and those of primary metabolism. These findings reveal an important example of molecular recognition between protein components that are essential for biosynthetic fidelity in natural product assembly and modification.
