Mycobacterium tuberculosis Maltosyltransferase GlgE, a Genetically Validated Antituberculosis Target, Is Negatively Regulated by Ser/Thr Phosphorylation

Abstract

GlgE is a maltosyltransferase involved in the biosynthesis of α-glucans that has been genetically validated as a potential therapeutic target against Mycobacterium tuberculosis. Despite also making α-glucan, the GlgC/GlgA glycogen pathway is distinct and allosterically regulated. We have used a combination of genetics and biochemistry to establish how the GlgE pathway is regulated. M. tuberculosis GlgE was phosphorylated specifically by the Ser/Thr protein kinase PknB in vitro on one serine and six threonine residues. Furthermore, GlgE was phosphorylated in vivo when expressed in Mycobacterium bovis bacillus Calmette–Guérin (BCG) but not when all seven phosphorylation sites were replaced by Ala residues. The GlgE orthologues from Mycobacterium smegmatis and Streptomyces coelicolor were phosphorylated by the corresponding PknB orthologues in vitro, implying that the phosphorylation of GlgE is widespread among actinomycetes. PknB-dependent phosphorylation of GlgE led to a 2 orders of magnitude reduction in catalytic efficiency in vitro. The activities of phosphoablative and phosphomimetic GlgE derivatives, where each phosphorylation site was substituted with either Ala or Asp residues, respectively, correlated with negative phosphoregulation. Complementation studies of a M. smegmatis glgE mutant strain with these GlgE derivatives, together with both classical and chemical forward genetics, were consistent with flux through the GlgE pathway being correlated with GlgE activity. We conclude that the GlgE pathway appears to be negatively regulated in actinomycetes through the phosphorylation of GlgE by PknB, a mechanism distinct from that known in the classical glycogen pathway. Thus, these findings open new opportunities to target the GlgE pathway therapeutically.

Genetics of Mycobacterial Trehalose Metabolism

“…In agreement with the toxicity of maltose-1-phosphate as the basis of essentiality of GlgE and GlgB, prevention of maltose-1-phosphate formation through genetic or pharmacological inactivation of TreS allows deletion of both glgE and glgB in M. tuberculosis (23). Recently, it was demonstrated that GlgE activity can be negatively fine-tuned via phosphorylation by the serine/ threonine protein kinase PknB (72), although it is unclear if this regulation is important for M. tuberculosis virulence. Given the high toxicity of maltose-1-phosphate, its synthesis rate also needs to be subject to one or more regulatory mechanisms in order to balance production and consumption.…”

Structural Insight into How Streptomyces coelicolor Maltosyl Transferase GlgE Binds α-Maltose 1-Phosphate and Forms a Maltosyl-enzyme Intermediate

“…3,10 To further explore their commonalities, the M. tuberculosis enzyme was exposed to 2-deoxy-2-fluoro-α-maltosyl fluoride. MALDI-TOF MS identified an increase in mass of the intact protein of 324 Da, from 80316 Da (80316 Da, expected) to 80640 Da, consistent with formation of a covalent intermediate despite it being the wild-type protein rather than an active site mutein.…”

Diversity of phosphorylases in glycoside hydrolase families

“…This is the only phosphorylase that is considered to be involved in the synthesis of glycoside. After the publication of this report, a series of reports considering the inhibitor of GlgE were published with the expectation that they may aid the development of a novel drug for multidrug-resistant strains of M. tuberculosis, the notorious pathogenic bacterium that causes tuberculosis (Kalscheuer et al 2010;Leiba et al 2013;Veleti et al 2014;Syson et al 2014;Sengupta et al 2014Sengupta et al , 2015. The 3D structure of the enzyme from Streptomyces coelicolor is available (Syson et al 2011).…”