Protein Acetylation in Pathogen Virulence and Host Defense: In Vitro Detection of Protein Acetylation by Radiolabeled Acetyl Coenzyme A

2019 ◽  
pp. 23-32
Author(s):  
Karl J. Schreiber ◽  
Jennifer D. Lewis
Keyword(s):  
Host Defense ◽  
Coenzyme A ◽  
Protein Acetylation ◽  
Acetyl Coenzyme A ◽  
Pathogen Virulence ◽  
Acetyl Coenzyme

scholarly journals In Bacillus subtilis, the Sirtuin Protein Deacetylase, Encoded by the srtN Gene (Formerly yhdZ), and Functions Encoded by the acuABC Genes Control the Activity of Acetyl Coenzyme A Synthetase

2009 ◽  
Vol 191 (6) ◽  
pp. 1749-1755 ◽  
Author(s):  
Jeffrey G. Gardner ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Bacillus Subtilis ◽  
Coenzyme A ◽  
Acetyl Coenzyme A ◽  
Acetyl Coenzyme ◽  
New Information ◽  
Low Concentrations ◽  
Dependent Protein ◽  
Protein Deacetylase

ABSTRACT This report provides in vivo evidence for the posttranslational control of the acetyl coenzyme A (Ac-CoA) synthetase (AcsA) enzyme of Bacillus subtilis by the acuA and acuC gene products. In addition, both in vivo and in vitro data presented support the conclusion that the yhdZ gene of B. subtilis encodes a NAD+-dependent protein deacetylase homologous to the yeast Sir2 protein (also known as sirtuin). On the basis of this new information, a change in gene nomenclature, from yhdZ to srtN (for sirtuin), is proposed to reflect the activity associated with the YdhZ protein. In vivo control of B. subtilis AcsA function required the combined activities of AcuC and SrtN. Inactivation of acuC or srtN resulted in slower growth and cell yield under low-acetate conditions than those of the wild-type strain, and the acuC srtN strain grew under low-acetate conditions as poorly as the acsA strain. Our interpretation of the latter result was that both deacetylases (AcuC and SrtN) are needed to maintain AcsA as active (i.e., deacetylated) so the cell can grow with low concentrations of acetate. Growth of an acuA acuC srtN strain on acetate was improved over that of the acuA + acuC srtN strain, indicating that the AcuA acetyltransferase enzyme modifies (i.e., inactivates) AcsA in vivo, a result consistent with previously reported in vitro evidence that AcsA is a substrate of AcuA.

scholarly journals Isolated Poly(3-Hydroxybutyrate) (PHB) Granules Are Complex Bacterial Organelles Catalyzing Formation of PHB from Acetyl Coenzyme A (CoA) and Degradation of PHB to Acetyl-CoA

2007 ◽  
Vol 189 (22) ◽  
pp. 8250-8256 ◽  
Author(s):  
Keiichi Uchino ◽  
Terumi Saito ◽  
Birgit Gebauer ◽  
Dieter Jendrossek
Keyword(s):  
Coenzyme A ◽  
Ralstonia Eutropha ◽  
Major Product ◽  
Acetyl Coa ◽  
Native Form ◽  
Acetyl Coenzyme A ◽  
Acetyl Coenzyme ◽  
Phb Synthase ◽  
Coa Reductase

ABSTRACTPoly(3-hydroxybutyrate) (PHB) granules isolated in native form (nPHB granules) fromRalstonia eutrophacatalyzed formation of PHB from14C-labeled acetyl coenzyme A (CoA) in the presence of NADPH and concomitantly released CoA, revealing that PHB biosynthetic proteins (acetoacetyl-CoA thiolase, acetoacetyl-CoA reductase, and PHB synthase) are present and active in isolated nPHB granules in vitro. nPHB granules also catalyzed thiolytic cleavage of PHB in the presence of added CoA, resulting in synthesis of 3-hydroxybutyryl-CoA (3HB-CoA) from PHB. Synthesis of 3HB-CoA was also shown by incubation of artificial (protein-free) PHB with CoA and PhaZa1, confirming that PhaZa1 is a PHB depolymerase catalyzing the thiolysis reaction. Acetyl-CoA was the major product detectable after incubation of nPHB granules in the presence of NAD+, indicating that downstream mobilizing enzyme activities were also present and active in isolated nPHB granules. We propose that intracellular concentrations of key metabolites (CoA, acetyl-CoA, 3HB-CoA, NAD+/NADH) determine whether a cell accumulates or degrades PHB. Since the degradation product of PHB is 3HB-CoA, the cells do not waste energy by synthesis and degradation of PHB. Thus, our results explain the frequent finding of simultaneous synthesis and breakdown of PHB.

scholarly journals Biochemical and Mutational Analyses of AcuA, the Acetyltransferase Enzyme That Controls the Activity of the Acetyl Coenzyme A Synthetase (AcsA) in Bacillus subtilis

2008 ◽  
Vol 190 (14) ◽  
pp. 5132-5136 ◽  
Author(s):  
Jeffrey G. Gardner ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Bacillus Subtilis ◽  
Coenzyme A ◽  
Effective Means ◽  
Wild Type ◽  
Modification System ◽  
Vitro Assay ◽  
Acetyl Coenzyme A ◽  
Acetyl Coenzyme

ABSTRACT The acuABC genes of Bacillus subtilis comprise a putative posttranslational modification system. The AcuA protein is a member of the Gcn5-related N-acetyltransferase (GNAT) superfamily, the AcuC protein is a class I histone deacetylase, and the role of the AcuB protein is not known. AcuA controls the activity of acetyl coenzyme A synthetase (AcsA; EC 6.2.1.1) in this bacterium by acetylating residue Lys549. Here we report the kinetic analysis of wild-type and variant AcuA proteins. We contrived a genetic scheme for the identification of AcuA residues critical for activity. Changes at residues H177 and G187 completely inactivated AcuA and led to its rapid turnover. Changes at residues R42 and T169 were less severe. In vitro assay conditions were optimized, and an effective means of inactivating the enzyme was found. The basic kinetic parameters of wild-type and variant AcuA proteins were obtained and compared to those of eukaryotic GNATs. Insights into how the isolated mutations may exert their deleterious effect were investigated by using the crystal structure of an AcuA homolog.

scholarly journals Presence of Acetyl Coenzyme A (CoA) Carboxylase and Propionyl-CoA Carboxylase in Autotrophic Crenarchaeota and Indication for Operation of a 3-Hydroxypropionate Cycle in Autotrophic Carbon Fixation

1999 ◽  
Vol 181 (4) ◽  
pp. 1088-1098 ◽  
Author(s):  
Castor Menendez ◽  
Zsuzsa Bauer ◽  
Harald Huber ◽  
Nasser Gad’on ◽  
Karl-Otto Stetter ◽  
...  
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
Carbon Fixation ◽  
Coenzyme A ◽  
Autotrophic Growth ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Carboxylase Activity ◽  
Acetyl Coenzyme A ◽  
Acetyl Coenzyme

ABSTRACT The pathway of autotrophic CO2 fixation was studied in the phototrophic bacterium Chloroflexus aurantiacus and in the aerobic thermoacidophilic archaeon Metallosphaera sedula. In both organisms, none of the key enzymes of the reductive pentose phosphate cycle, the reductive citric acid cycle, and the reductive acetyl coenzyme A (acetyl-CoA) pathway were detectable. However, cells contained the biotin-dependent acetyl-CoA carboxylase and propionyl-CoA carboxylase as well as phosphoenolpyruvate carboxylase. The specific enzyme activities of the carboxylases were high enough to explain the autotrophic growth rate via the 3-hydroxypropionate cycle. Extracts catalyzed the CO2-, MgATP-, and NADPH-dependent conversion of acetyl-CoA to 3-hydroxypropionate via malonyl-CoA and the conversion of this intermediate to succinate via propionyl-CoA. The labelled intermediates were detected in vitro with either 14CO2 or [14C]acetyl-CoA as precursor. These reactions are part of the 3-hydroxypropionate cycle, the autotrophic pathway proposed forC. aurantiacus. The investigation was extended to the autotrophic archaea Sulfolobus metallicus andAcidianus infernus, which showed acetyl-CoA and propionyl-CoA carboxylase activities in extracts of autotrophically grown cells. Acetyl-CoA carboxylase activity is unexpected in archaea since they do not contain fatty acids in their membranes. These aerobic archaea, as well as C. aurantiacus, were screened for biotin-containing proteins by the avidin-peroxidase test. They contained large amounts of a small biotin-carrying protein, which is most likely part of the acetyl-CoA and propionyl-CoA carboxylases. Other archaea reported to use one of the other known autotrophic pathways lacked such small biotin-containing proteins. These findings suggest that the aerobic autotrophic archaea M. sedula,S. metallicus, and A. infernus use a yet-to-be-defined 3-hydroxypropionate cycle for their autotrophic growth. Acetyl-CoA carboxylase and propionyl-CoA carboxylase are proposed to be the main CO2 fixation enzymes, and phosphoenolpyruvate carboxylase may have an anaplerotic function. The results also provide further support for the occurrence of the 3-hydroxypropionate cycle in C. aurantiacus.

scholarly journals ZmaR, a Novel and Widespread Antibiotic Resistance Determinant That Acetylates Zwittermicin A

1999 ◽  
Vol 181 (17) ◽  
pp. 5455-5460 ◽  
Author(s):  
Elizabeth A. Stohl ◽  
Sean F. Brady ◽  
Jon Clardy ◽  
Jo Handelsman
Keyword(s):  
Antibiotic Resistance ◽  
Coenzyme A ◽  
Sequence Similarity ◽  
Covalent Modification ◽  
Resistance Determinant ◽  
Acetyl Coenzyme A ◽  
Acetyl Coenzyme ◽  
Cell Extracts ◽  
Zwittermicin A

ABSTRACT ZmaR is a resistance determinant of unusual abundance in the environment and confers on gram-positive and gram-negative bacteria resistance to zwittermicin A, a novel broad-spectrum antibiotic produced by species of Bacillus. The ZmaR protein has no sequence similarity to proteins of known function; thus, the purpose of the present study was to determine the function of ZmaR in vitro. Cell extracts of E. coli containing zmaR inactivated zwittermicin A by covalent modification. Chemical analysis of inactivated zwittermicin A by 1H NMR, 13C NMR, and high- and low-resolution mass spectrometry demonstrated that the inactivated zwittermicin A was acetylated. Purified ZmaR protein inactivated zwittermicin A, and biochemical assays for acetyltransferase activity with [14C]acetyl coenzyme A demonstrated that ZmaR catalyzes the acetylation of zwittermicin A with acetyl coenzyme A as a donor group, suggesting that ZmaR may constitute a new class of acetyltransferases. Our results allow us to assign a biochemical function to a resistance protein that has no sequence similarity to proteins of known function, contributing fundamental knowledge to the fields of antibiotic resistance and protein function.

scholarly journals Antimalarial pantothenamide metabolites target acetyl–coenzyme A biosynthesis in Plasmodium falciparum

2019 ◽  
Vol 11 (510) ◽  
pp. eaas9917 ◽  
Author(s):  
Joost Schalkwijk ◽  
Erik L. Allman ◽  
Patrick A. M. Jansen ◽  
Laura E. de Vries ◽  
Julie M. J. Verhoef ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Coenzyme A ◽  
Mosquito Vector ◽  
Malaria Parasites ◽  
Humanized Mouse Model ◽  
Acetyl Coenzyme A ◽  
Block Transmission ◽  
Acetyl Coenzyme ◽  
Acyl Coenzyme A

Malaria eradication is critically dependent on new therapeutics that target resistant Plasmodium parasites and block transmission of the disease. Here, we report that pantothenamide bioisosteres were active against blood-stage Plasmodium falciparum parasites and also blocked transmission of sexual stages to the mosquito vector. These compounds were resistant to degradation by serum pantetheinases, showed favorable pharmacokinetic properties, and cleared parasites in a humanized mouse model of P. falciparum infection. Metabolomics revealed that coenzyme A biosynthetic enzymes converted pantothenamides into coenzyme A analogs that interfered with parasite acetyl–coenzyme A anabolism. Resistant parasites generated in vitro showed mutations in acetyl–coenzyme A synthetase and acyl–coenzyme A synthetase 11. Introduction and reversion of these mutations in P. falciparum using CRISPR-Cas9 gene editing confirmed the roles of these enzymes in the sensitivity of the malaria parasites to pantothenamides. These pantothenamide compounds with a new mode of action may have potential as drugs against malaria parasites.

Lipid metabolism in finishing bulls and steers implanted with oestradiol-17 β-dipropionate

1983 ◽  
Vol 37 (1) ◽  
pp. 81-88 ◽  
Author(s):  
R. L. Prior ◽  
S. B. Smith ◽  
B. D. Schanbacher ◽  
H. J. Mersmann
Keyword(s):  
Adipose Tissue ◽  
Coenzyme A ◽  
Experimental Treatment ◽  
Cell Number ◽  
Aconitate Hydratase ◽  
Acetyl Coenzyme A ◽  
Adipose Cell ◽  
Acetyl Coenzyme ◽  
Acetyl Coenzyme A Carboxylase

ABSTRACTTwenty bull calves were assigned to one of four treatment groups: 1) intact bulls; 2) intact bulls implanted with oestradiol dipropionate; 3) steers, and 4) steers implanted with oestradiol dipropionate. Subcutaneous adipose tissue was obtained by biopsy 5 months after implantation. Experimental treatment did not alter (P > 0·05) mean adipose cell volume, diameter or cell number per g adipose tissue. Soluble protein content of adipose cells from bulls was increased by implantation, but not in steers. In vitro rates of fatty acid synthesis were higher in oestrogen-implanted cattle compared with nonimplanted cattle when expressed per g adipose tissue, but not when expressed per cell. Maximal activities of the lipogenic-related enzymes, acetyl coenzyme A carboxylase, adenosine triphosphate-citrate lyase, aconitate hydratase and NADP-isocitrate dehydrogenase were higher in implanted compared with nonimplanted cattle when the activities were expressed per g tissue and acetyl coenzyme A carboxylase and aconitase were also higher when expressed per adipose cell. Bulls and steers had the same basal and stimulated lipolytic rates and implantation of oestradiol dipropionate had no effect on either basal or stimulated lipolytic rate. In this experiment, the oestrogenic implant appeared to have a stimulatory effect on in vitro estimates of lipogenesis.

scholarly journals PanM, an Acetyl-Coenzyme A Sensor Required for Maturation of l-Aspartate Decarboxylase (PanD)

mBio ◽  
2012 ◽  
Vol 3 (4) ◽  
Author(s):  
Tara N. Stuecker ◽  
Alex C. Tucker ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Coenzyme A ◽  
Lysine Acetylation ◽  
Acetyl Coa ◽  
Transfer Event ◽  
Acetyl Coenzyme A ◽  
Content Type ◽  
Acetyltransferase Activity ◽  
Acetyl Coenzyme

ABSTRACTCoenzyme A (CoA) is essential for cellular chemistry in all forms of life. The pantothenate moiety of CoA is generated from the condensation of pantoate and β-alanine. β-Alanine is formed by decarboxylation ofl-aspartate catalyzed by PanD, a pyruvoyl enzyme that is synthesized by the cell as an inactive precursor (pro-PanD). Maturation of pro-PanD into PanD occurs via a self-cleavage event at residue Ser25, which forms the catalytic pyruvoyl moiety. We recently reported thatSalmonella entericaPanM was necessary for pro-PanD maturation, bothin vitroandin vivo. Notably, PanM is annotated as a Gcn5-likeN-acetyltransferase (GNAT), which suggested that lysine acetylation might be part of the mechanism of maturation. Here we show that PanM lacks acetyltransferase activity and that acetyl-CoA stimulates its activity. Results of experiments with nonhydrolyzable ethyl-CoA and genetically encoded acetyl-lysine-containing PanD support the conclusion that PanM-dependent pro-PanD maturation does not involve an acetyl transfer event. We also show that CoA binding to PanM is needed forin vivoactivity and that disruption of CoA binding prevents PanM from interacting with PanD. We conclude that PanM is a GNAT homologue that lost its acetyltransferase activity and evolved a new function as an acetyl-CoA sensor that can trigger the maturation of pro-PanD.IMPORTANCENε-lysine acetylation is increasingly being recognized as a widespread and important form of posttranslational regulation in bacteria. The acetyltransferases that catalyze these reactions are poorly characterized in bacteria. Based on annotation, most bacterial genomes contain several acetyltransferases, but the physiological roles of only a handful have been determined. Notably, a subset of putative acetyltransferases lack residues that are critical for activity in most biochemically characterized acetyltransferases. We show that one such putative acetyltransferase, PanM (formerly YhhK), lacks acetyltransferase activity but functions instead as an acetyl-coenzyme A (CoA) sensor. This work establishes the possibility that, like PanM, other putative acetyltransferases may have evolved new functions while retaining the ability to sense acetyl-CoA.

Sign in / Sign up

Export Citation Format

Share Document