Biochemical characterization of the maltokinase from Mycobacterium bovis BCG

Abstract

BackgroundMaltose-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.ResultsThe 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 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.ConclusionsThe 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.

Genetics of Mycobacterial Trehalose Metabolism

“…Although the equilibrium of purified TreS favors the formation of trehalose from maltose in vitro, flux through TreS in vivo is in the opposite direction (24), which is driven by the rapid and irreversible ATP-dependent phosphorylation of the formed maltose to maltose-1-phosphate by the maltose kinase Pep2 (Rv0127) (26,27). The observed finding of the direction of flux through TreS for consumption of trehalose in M. smegmatis contradicts a previous study (9).…”

“…5), which is now known as the GlgE pathway and which is widespread among prokaryotes (23,25). TreS interconverts trehalose and maltose with formation of the α-anomer of maltose (24), which is subsequently rapidly and quantitatively phosphorylated in an ATPdependent reaction to maltose-1-phosphate by the maltose kinase Pep2 (Rv0127) (27). Maltose-1-phosphate then acts as the activated donor substrate for the key enzyme of this pathway, the maltosyltransferase GlgE (Rv1327c), which transfers the maltosyl moiety to the nonreducing 4′-hydroxyl group of an α-1,4-glucan acceptor molecule, yielding linear alpha-glucans.…”

Molecular fingerprints for a novel glucosamine kinase family inActinobacteria

“…In a few actinobacterial genomes a gene with sequence homology to maltokinases (and annotated as such) was identified, in addition to the canonical maltokinase gene (Mendes et al, 2010). In each species, the sequence identity between the canonical maltokinase and its paralogue was around 30%.…”

The cell envelope glycoconjugates ofMycobacterium tuberculosis

“…This four-step pathway generates α-(1,4)-glucans from trehalose and involves the trehalose synthase TreS, the maltokinase Pep2, the maltose-1-phosphate maltosyltransferase GlgE, and GlgB [Fig. 14] (Pan et al ., 2004; Pan et al ., 2008; Kalscheuer et al ., 2010, Elbein et al ., 2010; Mendes et al ., 2010; Miah et al ., 2013). Disruption of glgE , like that of glgB , results in the accumulation of maltose-1-phosphate which is toxic to the cells.…”