Degree Type

Dissertation

Date of Award

2019

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

Major

Biochemistry

First Advisor

Basil J. Nikolau

Second Advisor

Marna D. Yandeau-Nelson

Abstract

As an activated form of acetate, acetyl-CoA is a key metabolic intermediate that links many metabolic processes, including the TCA cycle, amino acid metabolism, fatty acid metabolism and biosynthetic processes that generate many polyketides and some terpenes. This molecule is also involved in regulation of proteins, through the post translational modification of reversible acetylation. It is the most prevalent thioester-containing intermediate of metabolism, and biological systems utilize multiple enzymatic mechanisms to generate this central metabolite. Acetyl-CoA synthetase (ACS) is a member of a large super-family of enzymes that display diverse substrate specificities, with a common mechanism of catalyzing the formation of a thioester bond between Coenzyme A and a carboxylic acid, while hydrolyzing ATP to AMP and pyrophosphate. We explored the structural basis of the substrate specificity of ACS which specifically makes acetyl-CoA. This enzyme was redesigned successfully, which switched a highly specific enzyme from using only acetate, to be equally specific for using longer linear (up to hexanoate) or branched chain (methylvalerate) carboxylate substrates.

ACS is known to be regulated through the post translational modification of reversible acetylation. In organisms like bacteria and humans, it has been shown that this modification leads to reduction of activity of the enzyme. In this study we have analyzed the effect of acetylation on Arabidopsis ACS activity and the conserved lysine involved in this modification. This study shows that effect of acetylation on this plant enzyme is very different from what is observed in homologous ACS. According to our knowledge, this is the first report of how acetylation affects a plant ACS, using in vitro studies.

Sirtuins are histone deacetylases involved in deacetylating other proteins and thus involved in epigenetic and metabolic regulation. We characterized and compared Arabidopsis sirtuins, SRT1 and SRT2 both in vitro and in vivo, through total untargeted metabolomics, acetylome analysis and through heterologous protein expression and characterization. A comparative integrated biochemical-genetic study of the acetylome and metabolome of srt1 or srt2 single mutants and srt1srt2 double mutant plants indicate that although there are undetectable effects on the growth phenotype of the plants, there are considerable changes in the molecular features that were assayed. The changes induced by the double mutant indicate that these proteins deacetylate both histone and non-histone proteins. In addition, the mutant-induced changes in the metabolome indicate that the two SRT homologs are involved in regulating different metabolic processes, generating distinct metabolomes. This study also explores the effect of these two deacetylases on Arabidopsis ACS acetylation. In summary the study of the proteins, acetyl-CoA synthetase and sirtuins in Arabidopsis, will help to further the understanding of acetyl-CoA metabolism in plants.

Copyright Owner

Naazneen Sofeo

Language

en

File Format

application/pdf

File Size

139 pages

Available for download on Friday, March 12, 2021

Included in

Biochemistry Commons

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