Biochemical characterization of the maltokinase from Mycobacterium bovis BCG

Abstract

<p>Abstract</p> <p>Background</p> <p>Maltose-1-phosphate was detected in <it>Mycobacterium bovis </it>BCG extracts in the 1960's but a maltose-1-phosphate synthetase (maltokinase, Mak) was only much later purified from <it>Actinoplanes missouriensis</it>, allowing the identification of the <it>mak </it>gene. Recently, this metabolite was proposed to be the intermediate in a pathway linking trehalose with the synthesis of glycogen in <it>M. smegmatis</it>. Although the <it>M. tuberculosis </it>H37Rv <it>mak </it>gene (Rv0127) was considered essential for growth, no mycobacterial Mak has, to date, been characterized.</p> <p>Results</p> <p>The sequence of the Mak from <it>M. bovis </it>BCG was identical to that from <it>M. tuberculosis </it>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 K_{<it>m </it>}for maltose was 2.52 ± 0.40 mM and 0.74 ± 0.12 mM for ATP; the <it>V</it>_maxwas 21.05 ± 0.89 μmol/min.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°C, between pH 7 and 9 (at 37°C) and was unstable on ice and upon freeze/thawing. The addition of 50 mM NaCl markedly enhanced Mak stability.</p> <p>Conclusions</p> <p>The unknown role of maltokinases in mycobacterial metabolism and the lack of biochemical data led us to express the <it>mak </it>gene from <it>M. bovis </it>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 <it>M. tuberculosis</it>, its high-level production and purification in bioactive form provide important tools for further functional and structural studies.</p