Abstract
In bacteria, glucosamine-6-phosphate (GlcN6P) synthase, GlmS, is an enzyme required for the synthesis of Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a precursor of peptidoglycan. In Bacillus subtilis, an UDP-GlcNAc binding protein, GlmR (formerly YvcK), essential for growth on non-glycolytic carbon sources, has been proposed to stimulate GlmS activity; this activation could be antagonized by UDP-GlcNAc. Using purified proteins, we demonstrate that GlmR directly stimulates GlmS activity and the presence of UDP-GlcNAc (at concentrations above 0.1 mM) prevents this regulation. We also showed that YvcJ, whose gene is associated with yvcK (glmR), interacts with GlmR in an UDP-GlcNAc dependent manner. Strains producing GlmR variants unable to interact with YvcJ show decreased transformation efficiency similar to that of a yvcJ null mutant. We therefore propose that, depending on the intracellular concentration of UDP-GlcNAc, GlmR interacts with either YvcJ or GlmS. When UDP-GlcNAc concentration is high, this UDP-sugar binds to YvcJ and to GlmR, blocking the stimulation of GlmS activity and driving the interaction between GlmR and YvcJ to probably regulate the cellular role of the latter. When the UDP-GlcNAc level is low, GlmR does not interact with YvcJ and thus does not regulate its cellular role but interacts with GlmS to stimulate its activity.
Highlights
Abbreviations GlcN6P Glucosamine-6-phosphate PG Peptidoglycan UDP-GlcNAc Uridine diphosphate N-acetylglucosamine fructose 6-phosphate (F6P) Fructose 6-phosphate Gln Glutamine GlcN6P Glucosamine-6-phosphate GNA-1 GlcN6P N-acetyltransferase 1 HPAEC-PAD High Pressure Anion Exchange Chromatography coupled with Pulsed Amperometric
Using purified proteins and measuring the production of GlcN6P, we showed that GlmR directly regulates GlmS activity and this stimulatory effect is inhibited by UDP-GlcNAc
Patel et al suggested that GlmR activates GlmS in B. subtilis, probably directly since the two proteins interact by a bacterial two-hybrid assay[9]
Summary
Abbreviations GlcN6P Glucosamine-6-phosphate PG Peptidoglycan UDP-GlcNAc Uridine diphosphate N-acetylglucosamine F6P Fructose 6-phosphate Gln Glutamine GlcN6P Glucosamine-6-phosphate GNA-1 GlcN6P N-acetyltransferase 1 HPAEC-PAD High Pressure Anion Exchange Chromatography coupled with Pulsed Amperometric. These results show that UDP-GlcNAc controls the cellular role of GlmR and its interaction with either YvcJ or GlmS. In the presence of 1 mM of UDP-GlcNAc, as expected, we did not detect the GlmR-stimulatory effect on GlmS activity (Fig. 1B).
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