Karl Syson1, Sibyl F D Batey2, Steffen Schindler3, Rainer Kalscheuer4, Stephen Bornemann5. 1. Biological Chemistry Department, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom. Electronic address: karl.syson@astrazeneca.com. 2. Biological Chemistry Department, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom. Electronic address: Sibyl.batey@jic.ac.uk. 3. Institute of Pharmaceutical Biology and Biotechnology, Heinrich Heine University, 40225 Düsseldorf, Germany. Electronic address: Steffen.schindler@hhu.de. 4. Institute of Pharmaceutical Biology and Biotechnology, Heinrich Heine University, 40225 Düsseldorf, Germany. Electronic address: Rainer.Kalscheuer@hhu.de. 5. Biological Chemistry Department, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom. Electronic address: stephen.bornemann@tsl.ac.uk.
Abstract
BACKGROUND: The bacterial GlgE pathway is the third known route to glycogen and is the only one present in mycobacteria. It contributes to the virulence of Mycobacterium tuberculosis. The involvement of GlgE in glycogen biosynthesis was discovered twenty years ago when the phenotype of a temperature-sensitive Mycobacterium smegmatis mutation was rescued by the glgE gene. The evidence at the time suggested glgE coded for a glucanase responsible for the hydrolysis of glycogen, in stark contrast with recent evidence showing GlgE to be a polymerase responsible for its biosynthesis. METHODS: We reconstructed and examined the temperature-sensitive mutant and characterised the mutated GlgE enzyme. RESULTS: The mutant strain accumulated the substrate for GlgE, α-maltose-1-phosphate, at the non-permissive temperature. The glycogen assay used in the original study was shown to give a false positive result with α-maltose-1-phosphate. The accumulation of α-maltose-1-phosphate was due to the lowering of the kcat of GlgE as well as a loss of stability 42 °C. The reported rescue of the phenotype by GarA could potentially involve an interaction with GlgE, but none was detected. CONCLUSIONS: We have been able to reconcile apparently contradictory observations and shed light on the basis for the phenotype of the temperature-sensitive mutation. GENERAL SIGNIFICANCE: This study highlights how the lowering of flux through the GlgE pathway can slow the growth mycobacteria.
BACKGROUND: The bacterial GlgE pathway is the third known route to glycogen and is the only one present in mycobacteria. It contributes to the virulence of Mycobacterium tuberculosis. The involvement of GlgE in glycogen biosynthesis was discovered twenty years ago when the phenotype of a temperature-sensitive Mycobacterium smegmatis mutation was rescued by the glgE gene. The evidence at the time suggested glgE coded for a glucanase responsible for the hydrolysis of glycogen, in stark contrast with recent evidence showing GlgE to be a polymerase responsible for its biosynthesis. METHODS: We reconstructed and examined the temperature-sensitive mutant and characterised the mutated GlgE enzyme. RESULTS: The mutant strain accumulated the substrate for GlgE, α-maltose-1-phosphate, at the non-permissive temperature. The glycogen assay used in the original study was shown to give a false positive result with α-maltose-1-phosphate. The accumulation of α-maltose-1-phosphate was due to the lowering of the kcat of GlgE as well as a loss of stability 42 °C. The reported rescue of the phenotype by GarA could potentially involve an interaction with GlgE, but none was detected. CONCLUSIONS: We have been able to reconcile apparently contradictory observations and shed light on the basis for the phenotype of the temperature-sensitive mutation. GENERAL SIGNIFICANCE: This study highlights how the lowering of flux through the GlgE pathway can slow the growth mycobacteria.
Authors: Patrick England; Annemarie Wehenkel; Sonia Martins; Sylviane Hoos; Gwénaëlle André-Leroux; Andrea Villarino; Pedro M Alzari Journal: FEBS Lett Date: 2008-12-27 Impact factor: 4.124
Authors: Abdul M Rashid; Sibyl F D Batey; Karl Syson; Hendrik Koliwer-Brandl; Farzana Miah; J Elaine Barclay; Kim C Findlay; Karol P Nartowski; Yaroslav Z Khimyak; Rainer Kalscheuer; Stephen Bornemann Journal: Biochemistry Date: 2016-06-02 Impact factor: 3.162
Authors: Karl Syson; Clare E M Stevenson; David M Lawson; Stephen Bornemann Journal: Acta Crystallogr F Struct Biol Commun Date: 2020-04-03 Impact factor: 1.056