title: 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.
authors.affiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Universités de Montpellier II et I, CNRS, UMR 5235, case 107, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France.
authors.forename: Jade
authors.initials: J
authors.lastname: Leiba
authors.affiliation: Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom.
authors.forename: Karl
authors.initials: K
authors.lastname: Syson
authors.affiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Universités de Montpellier II et I, CNRS, UMR 5235, case 107, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France.
authors.forename: Grégory
authors.initials: G
authors.lastname: Baronian
authors.affiliation: Institut de Biologie et Chimie des Protéines (IBCP UMR 5086), CNRS, Université Lyon1, IFR128 BioSciences, Lyon Gerland, 7 passage du Vercors, 69367 Lyon Cedex 07, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Zanella-Cléon
authors.affiliation: Institute for Medical Microbiology and Hospital Hygiene, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Kalscheuer
authors.affiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Universités de Montpellier II et I, CNRS, UMR 5235, case 107, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France; INSERM, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Kremer
authors.affiliation: Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom. Electronic address: stephen.bornemann@jic.ac.uk.
authors.forename: Stephen
authors.initials: S
authors.lastname: Bornemann
authors.affiliation: Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Universités de Montpellier II et I, CNRS, UMR 5235, case 107, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France. Electronic address: virginie.molle@univ-montp2.fr.
authors.forename: Virginie
authors.initials: V
authors.lastname: Molle
mesh_terms: D000995:Antitubercular Agents; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D005816:Genetic Complementation Test; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D009163:Mycobacterium bovis / Q000201:enzymology / Q000235:genetics; D020102:Mycobacterium smegmatis / Q000201:enzymology / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D010766:Phosphorylation / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D048372:Streptomyces coelicolor / Q000201:enzymology / Q000235:genetics
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D005964:Glucosyltransferases; C436710:maltosyltransferase; C475060:PknB protein, Mycobacterium tuberculosis; D017346:Protein Serine-Threonine Kinases
keywords: Bacterial pathogenesis; Bacterial protein kinases; Microbiology; Mycobacterium tuberculosis; Serine threonine protein kinase; glucan; maltosyltransferase; phosphorylation