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Int J Mycobacteriol. 2018 Oct-Dec;7(4):368-374. doi: 10.4103/ijmy.ijmy_116_18.

Expression, purification, and characterization of pyruvate kinase from Mycobacterium tuberculosis: A key allosteric regulatory enzyme.

Author information

1
Department of Biotechnology, Kuvempu University, Shimoga, Karnataka; Department of Biotechnology, Vignan's Foundation for Science, Technology and Research (Deemed to be University), Vadlamudi, Guntur (Dt), Andra Pradesh, India.
2
Department of Biotechnology, Gulbarga University, Gulbarga, Karnataka, India.
3
Department of Biochemistry and Molecular Biology, Pondicherry University, Puducherry, India.
4
Department of Biotechnology, Kuvempu University, Shimoga, Karnataka, India.

Abstract

Background:

The current research aims to isolate pyruvate kinase (Pyk) gene from Mycobacterium tuberculosis and expression of the gene (Rv1617) to obtain a purified enzyme. The enzyme activity and secondary structural features were assessed through biochemical assays and circular dichroism (CD) spectroscopy, respectively.

Methods:

The Pyk-encoding gene from the complete genome of M. tuberculosis was cloned, sequenced, and expressed in Escherichia coli BL21 (DE3). The enzyme was purified by nickel-nitrilotriacetic acid affinity chromatography and enzyme activity was determined by a lactose dehydrogenase-coupled assay system. Further, far ultraviolet CD spectra of the enzyme and the substrate bound enzyme were analyzed using a Jasco J712 spectrophotometer.

Results:

A single protein with an approximate molecular mass of 54 kDa was purified and a specific activity of 5.31 units/mg was determined from purified M. tuberculosis Pyk. The activity of the enzyme indicating a protein is defined by separate domain for each catalytic function. The secondary structure analysis of CD spectra of the recombinant Pyk has revealed a content of 17% α-helix, 34% β-sheet, and 49% turns in the enzyme.

Conclusion:

The growing evidence has impacted M. tuberculosis central carbon metabolism as a key determinant of the survival and pathogenicity in the host. The purified Pyk was observed to have increased enzyme activity in all steps of purification. Retention of Pyk activity indicates a possible catalytic role for the lower part of the glycolytic pathway. The overall results of the spectra obtained from the CD suggest that the substrate phosphoenolpyruvate and adenosine diphosphate binding to the enzyme can cause conformational changes resulting in the exposure or shielding of residues susceptible to modification.

KEYWORDS:

Allosteric regulation; Mycobacterium tuberculosis; bacterial metabolism; circular dichroism; pathogenesis; pyruvate kinase

PMID:
30531037
DOI:
10.4103/ijmy.ijmy_116_18
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