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Structural and Functional Studies of Phosphoenolpyruvate Carboxykinase from Mycobacterium tuberculosis

SYSNO ASEP	0444002
Document Type	J - Journal Article
R&D Document Type	Journal Article
Subsidiary J	Článek ve WOS
Title	Structural and Functional Studies of Phosphoenolpyruvate Carboxykinase from Mycobacterium tuberculosis
Author(s)	Machová, Iva (UOCHB-X)_{RID, ORCID} Snášel, Jan (UOCHB-X)_RID Dostál, Jiří (UOCHB-X)_{RID, ORCID} Brynda, Jiří (UOCHB-X)_{RID, ORCID} Fanfrlík, Jindřich (UOCHB-X)_{RID, ORCID} Singh, M. (DE) Tarábek, Ján (UOCHB-X)_{RID, ORCID} Vaněk, O. (CZ) Bednárová, Lucie (UOCHB-X)_{RID, ORCID} Pichová, Iva (UOCHB-X)_{RID, ORCID}
Number of authors	10
Source Title	PLoS ONE. - : Public Library of Science - ISSN 1932-6203 Roč. 10, č. 3 (2015), e0120682/1-e0120682/21
Number of pages	21 s.
Publication form	Online - E
Language	eng - English
Country	US - United States
Keywords	crystal structure ; noncovalent complexes ; Mycobacterium tuberculosis ; mechanism
Subject RIV	CE - Biochemistry
R&D Projects	LO1302 GA MŠMT - Ministry of Education, Youth and Sports (MEYS)
Institutional support	UOCHB-X - RVO:61388963
UT WOS	000351987300164
EID SCOPUS	84925740525
DOI	10.1371/journal.pone.0120682
Annotation	Tuberculosis, the second leading infectious disease killer after HIV, remains a top public health priority. The causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), which can cause both acute and clinically latent infections, reprograms metabolism in response to the host niche. Phosphoenolpyruvate carboxykinase (Pck) is the enzyme at the center of the phosphoenolpyruvate-pyruvate-oxaloacetate node, which is involved in regulating the carbon flow distribution to catabolism, anabolism, or respiration in different states of Mtb infection. Under standard growth conditions, Mtb Pck is associated with gluconeogenesis and catalyzes the metal-dependent formation of phosphoenolpyruvate. In non-replicating Mtb, Pck can catalyze anaplerotic biosynthesis of oxaloacetate. Here, we present insights into the regulation of Mtb Pck activity by divalent cations. Through analysis of the Xray structure of Pck-GDP and Pck-GDP-Mn2+ complexes, mutational analysis of the GDP binding site, and quantum mechanical (QM)-based analysis, we explored the structural determinants of efficient Mtb Pck catalysis. We demonstrate that Mtb Pck requires presence of Mn2+ and Mg2+ cations for efficient catalysis of gluconeogenic and anaplerotic reactions. The anaplerotic reaction, which preferably functions in reducing conditions that are characteristic for slowed or stopped Mtb replication, is also effectively activated by Fe2+ in the presence of Mn2+ or Mg2+ cations. In contrast, simultaneous presence of Fe2+ and Mn2+ or Mg2+ inhibits the gluconeogenic reaction. These results suggest that inorganic ions can contribute to regulation of central carbon metabolism by influencing the activity of Pck. Furthermore, the X-ray structure determination, biochemical characterization, and QM analysis of Pck mutants confirmed the important role of the Phe triad for proper binding of the GDP-Mn2+ complex in the nucleotide binding site and efficient catalysis of the anaplerotic reaction.
Workplace	Institute of Organic Chemistry and Biochemistry
Contact	asep@uochb.cas.cz ; Kateřina Šperková, Tel.: 232 002 584 ; Viktorie Chládková, Tel.: 232 002 434
Year of Publishing	2016
Electronic address	http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0120682

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