Purification and characterization of the acetyl-CoA synthetase from Mycobacterium tuberculosis.

Abstract

Acetyl-CoA (AcCoA) synthetase (Acs) catalyzes the conversion of acetate into AcCoA, which is involved in many catabolic and anabolic pathways. Although this enzyme has been studied for many years in many organisms, the properties of Mycobacterium tuberculosis Acs and the regulation of its activity remain unknown. Here, the putative acs gene of M. tuberculosis H37Rv (Mt-Acs) was expressed as a fusion protein with 6×His-tag on the C-terminus in Escherichia coli. The recombinant Mt-Acs protein was successfully purified and then its enzymatic characteristics were analyzed. The optimal pH and temperature, and the kinetic parameters of Mt-Acs were determined. To investigate whether Mt-Acs is regulated by lysine acetylation as reported for Salmonella enterica Acs, its mutant K617R was also generated. Determination of the enzymatic activity suggests that Lys-617 is critical for its function. We further demonstrated that Mt-Acs underwent auto-acetylation with acetate but not with AcCoA as the acetyl donor, which resulted in the decrease of its activity. CoA, the substrate for AcCoA formation, inhibited the auto-acetylation. Furthermore, the silent information regulator (Sir2) of M. tuberculosis (Mt-Sir2) could catalyze Mt-Acs deacetylation, which resulted in activation of Acs. These results may provide more insights into the physiological roles of Mt-Acs in M. tuberculosis central metabolism.

• Authors: Ru Li, Jing Gu, Peng Chen, Zhiping Zhang, Jiaoyu Deng, Xianen Zhang
• Journal: Acta biochimica et biophysica Sinica (Acta Biochim Biophys Sin (Shanghai)) Vol. 43 Issue 11 Pg. 891-9 (Nov 2011) ISSN: 1745-7270 [Electronic] China
• PMID: 21896569 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Acetates
• Bacterial Proteins
• Acetyl Coenzyme A
• Hydrolases
• Sirtuins
• HsaD protein, Mycobacterium tuberculosis
• Acetate-CoA Ligase

Topics

• Acetate-CoA Ligase (genetics, isolation & purification, metabolism)
• Acetates (chemistry, metabolism)
• Acetyl Coenzyme A (metabolism)
• Acetylation
• Amino Acid Sequence
• Bacterial Proteins (genetics, isolation & purification, metabolism)
• Hydrolases (genetics, isolation & purification, metabolism)
• Molecular Sequence Data
• Mutation (genetics)
• Mycobacterium tuberculosis (enzymology)
• Sirtuins (genetics, metabolism)
• Substrate Specificity