Biochemical characterization of the maltokinase from Mycobacterium bovis BCG.
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.
Mendes
Vítor
V
Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal.
Maranha
Ana
A
Lamosa
Pedro
P
da Costa
Milton S
MS
Empadinhas
Nuno
N
D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D009163:Mycobacterium bovis / Q000201:enzymology*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013403:Sugar Phosphates / Q000378:metabolism; D013696:Temperature
D011994:Recombinant Proteins; D013403:Sugar Phosphates; C080751:maltose 1-phosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C100489:maltokinase; D008274:Magnesium