Dataset Information

Threonine 57 is required for the post-translational activation of Escherichia coli aspartate ?-decarboxylase.

ABSTRACT: Aspartate ?-decarboxylase is a pyruvoyl-dependent decarboxylase required for the production of ?-alanine in the bacterial pantothenate (vitamin B5) biosynthesis pathway. The pyruvoyl group is formed via the intramolecular rearrangement of a serine residue to generate a backbone ester intermediate which is cleaved to generate an N-terminal pyruvoyl group. Site-directed mutagenesis of residues adjacent to the active site, including Tyr22, Thr57 and Tyr58, reveals that only mutation of Thr57 leads to changes in the degree of post-translational activation. The crystal structure of the site-directed mutant T57V is consistent with a non-rearranged backbone, supporting the hypothesis that Thr57 is required for the formation of the ester intermediate in activation.

SUBMITTER: Webb ME

PROVIDER: S-EPMC3975893 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Threonine 57 is required for the post-translational activation of Escherichia coli aspartate α-decarboxylase.

Webb Michael E ME Yorke Briony A BA Kershaw Tom T Lovelock Sarah S Lobley Carina M C CM Kilkenny Mairi L ML Smith Alison G AG Blundell Tom L TL Pearson Arwen R AR Abell Chris C

Acta crystallographica. Section D, Biological crystallography 20140321 Pt 4

Aspartate α-decarboxylase is a pyruvoyl-dependent decarboxylase required for the production of β-alanine in the bacterial pantothenate (vitamin B5) biosynthesis pathway. The pyruvoyl group is formed via the intramolecular rearrangement of a serine residue to generate a backbone ester intermediate which is cleaved to generate an N-terminal pyruvoyl group. Site-directed mutagenesis of residues adjacent to the active site, including Tyr22, Thr57 and Tyr58, reveals that only mutation of Thr57 leads ...[more]

PMID: 24699660

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Project description:Enzymes catalyze a particular reaction in cells, but only a few control the rate of this reaction and the metabolic pathway that follows. One specific mechanism for such enzymatic control of a metabolic pathway involves molecular feedback, whereby a metabolite further down the pathway acts at a unique site on the control enzyme to alter its activity allosterically. This regulation may be positive or negative (or both), depending upon the particular system. Another method of enzymatic control involves the cooperative binding of the substrate, which allows a large change in enzyme activity to emanate from only a small change in substrate concentration. Allosteric regulation and homotropic cooperativity are often known to involve significant conformational changes in the structure of the protein. Escherichia coli aspartate transcarbamoylase (ATCase) is the textbook example of an enzyme that regulates a metabolic pathway, namely, pyrimidine nucleotide biosynthesis, by feedback control and by the cooperative binding of the substrate, L-aspartate. The catalytic and regulatory mechanisms of this enzyme have been extensively studied. A series of X-ray crystal structures of the enzyme in the presence and absence of substrates, products, and analogues have provided details, at the molecular level, of the conformational changes that the enzyme undergoes as it shifts between its low-activity, low-affinity form (T state) to its high-activity, high-affinity form (R state). These structural data provide insights into not only how this enzyme catalyzes the reaction between l-aspartate and carbamoyl phosphate to form N-carbamoyl-L-aspartate and inorganic phosphate, but also how the allosteric effectors modulate this activity. In this Account, we summarize studies on the structure of the enzyme and describe how these structural data provide insights into the catalytic and regulatory mechanisms of the enzyme. The ATCase-catalyzed reaction is regulated by nucleotide binding some 60 Å from the active site, inducing structural alterations that modulate catalytic activity. The delineation of the structure and function in this particular model system will help in understanding the molecular basis of cooperativity and allosteric regulation in other systems as well.

Project description:UNLABELLED:Coenzyme 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 ?-alanine. ?-Alanine is formed by decarboxylation of l-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 that Salmonella enterica PanM was necessary for pro-PanD maturation, both in vitro and in vivo. Notably, PanM is annotated as a Gcn5-like N-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 for in vivo activity 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. IMPORTANCE:N?-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.

Dataset Information

Threonine 57 is required for the post-translational activation of Escherichia coli aspartate ?-decarboxylase.

Publications

Threonine 57 is required for the post-translational activation of Escherichia coli aspartate α-decarboxylase.

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