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. 2010 May 27;11:21.
doi: 10.1186/1471-2091-11-21.

Biochemical Characterization of the Maltokinase From Mycobacterium Bovis BCG

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Free PMC article

Biochemical Characterization of the Maltokinase From Mycobacterium Bovis BCG

Vítor Mendes et al. BMC Biochem. .
Free PMC article

Abstract

Background: Maltose-1-phosphate was detected in Mycobacterium bovis BCG extracts in the 1960's but a maltose-1-phosphate synthetase (maltokinase, Mak) was only much later purified from Actinoplanes missouriensis, allowing the identification of the mak gene. Recently, this metabolite was proposed to be the intermediate in a pathway linking trehalose with the synthesis of glycogen in M. smegmatis. Although the M. tuberculosis H37Rv mak gene (Rv0127) was considered essential for growth, no mycobacterial Mak has, to date, been characterized.

Results: The sequence of the Mak from M. bovis BCG was identical to that from M. tuberculosis strains (99-100% amino acid identity). The enzyme was dependent on maltose and ATP, although GTP and UTP could be used to produce maltose-1-phosphate, which we identified by TLC and characterized by NMR. The Km for maltose was 2.52 +/- 0.40 mM and 0.74 +/- 0.12 mM for ATP; the Vmax was 21.05 +/- 0.89 micromol/min x mg(-1). Divalent cations were required for activity and Mg2+ was the best activator. The enzyme was a monomer in solution, had maximal activity at 60 degrees C, between pH 7 and 9 (at 37 degrees C) and was unstable on ice and upon freeze/thawing. The addition of 50 mM NaCl markedly enhanced Mak stability.

Conclusions: The unknown role of maltokinases in mycobacterial metabolism and the lack of biochemical data led us to express the mak gene from M. bovis BCG for biochemical characterization. This is the first mycobacterial Mak to be characterized and its properties represent essential knowledge towards deeper understanding of mycobacterial physiology. Since Mak may be a potential drug target in M. tuberculosis, its high-level production and purification in bioactive form provide important tools for further functional and structural studies.

Figures

Figure 1
Figure 1
Genetic environment of the mak gene in different organisms. Organization of the region containing the mak gene in Mycobacterium bovis BCG, M. tuberculosis, M. marinum, M. leprae, Actinoplanes missouriensis, Rubrobacter xylanophilus, Chloroflexus sp. and Pseudomonas entomophila. Arrows represent genes and their orientation. pepA, serine protease; treS, trehalose synthase; mak, maltokinase; glgB, glycogen branching enzyme; fused treS/mak, probable fused trehalose synthase/maltokinase.
Figure 2
Figure 2
SDS-PAGE showing the recombinant maltokinase (Mak) from M. bovis BCG. Lane 1 - Purified recombinant Mak. M - Protein molecular weight markers.
Figure 3
Figure 3
Kinetic studies of Mak activity. Dependence of Mak activity on the concentrations of maltose (A), ATP (B), GTP (C) and UTP (D).
Figure 4
Figure 4
Effects of temperature, pH and Mg2+ concentration on Mak activity. Temperature profile (A) and pH dependence (B) using ATP (white circle), GTP (black circle) and UTP (black triangle) as phosphate donors. (C) Effect of Mg2+ concentration.
Figure 5
Figure 5
1H-NMR spectra of the Mak reaction. Mixture contained 5 mM each ATP and maltose, 5 mM MgCl2 in 10 mM BTP at pH 8, prior (A) and after (B) the addition of 15 μg of Mak and incubation for 10 min at 37°C. The top panel (C) represents the structures of maltose and maltose-1-phosphate and is labeled according to their assigned resonances.
Figure 6
Figure 6
Metabolic circuit between trehalose, maltose and glycogen and proposal of alternative roles for maltose-1-phosphate. Data adapted from references [13,20,21]. Mak, maltokinase, TreS, trehalose synthase, TreY/TreZ, maltooligosyltrehalose synthase/trehalohydrolase, GMPMT, α-1,4-glucan: maltose-1-P maltosyltransferase.

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