Glucosamine-1P as a substrate in ADP-glucose pyrophosphorylases from Gram-positive bacteria

IGLESIAS MJ; IGLESIAS AA; ASENCION DIEZ MD

virtual

Congreso; LVII SAIB Meeting - XVI SAMIGE Meeting; 2021

The study of pyrophosphorylases (PPase) determiningthe hexose-1P fate in carbohydrate metabolism is critical for a deeperunderstanding regarding the use of microorganisms and their enzymes asbiotechnological tools. PPases “activate” glycosidic moieties in their form of sugar-1Pto an NDP-sugar, by means of NTP. The amino sugar glucosamine (GlcN) is anessential component of glycosaminoglycans and constitutes a glycosidic unit inmany antibiotics. GlcN-1P was only described as an intermediary in the pathwayfrom GlcN-6P to UDP-N-acetyl-GlcN. However, we recently described an PPasefrom Rhodococcus jostti capable of specifically catalyzing GlcN-1P andUTP to putatively synthesize UDP-GlcN. In addition, we found that ADP-glucosePPases (EC 2.7.7.27, ADPGlcPPase) from two Rhodococcus species were ableto use GlcN-1P alternatively to glucose-1P, their canonical substrate.Remarkably, the activity with GlcN-1P in the rhodococcal ADPGlcPPases wassensitive to allosteric regulators.We then extended the analysis of GlcN-1P consumption to otherADPGlcPPases from Gram-positive organisms, either Actinobacteria (genomic highG+C content) and Firmicutes (low G+C content). Is worthy to mention that thelatter group present ADPGlcPPases composed by two subunits: GlgC (catalyticallyactive) and GlgD (inactive). Then, the GlgC and GlgC/GlgD isoforms from Geobacillusstearothemophillus (Gst) and Ruminococcus albus (Ral)were analyzed regarding their ability to use GlcN-1P. In this regard, thehomotetrameric RalGlgC showed a 19.3% activity compared to glucose-1P,while for the heteromeric RalGlgC/GlgD was as low as 1%. The activitywith GlcN-1P in GstGlgC and GstGlgC/GlgD was 1.7% and 5%regarding glucose-1P. The specific enzyme activity using GlcN-1P for RalGlgCand GstGlgC/GlgD was 0.16 and 0.48 U/mg, which are one order ofmagnitude higher than the activity in the characterized rhodococcal enzyme. Inthe case of Actinobacteria, we studied the ADPGlcPPase from Kocuriarhizophila, a biotechnological important organism. The enzyme showed low activitywith GlcN-1P (0.03 U/mg) which was increased about 650-fold (up to 13.71 U/mg)in presence of 1 mM GlcN-6P, one of its main activators. The K. rhizophilaADPGlcPPase activity is in the same order of magnitude than the specific PPasefrom R. jostii, thus constituting important enzymological tools tosynthesize novel metabolites such as ADP-GlcN and UDP-GlcN, respectively. Thiswork supports a scenario for new molecules discovery based in alternatives forcarbohydrates metabolism and new tools for precision synthesis of innovativesugar compounds. Currently, we are designing a set-up of biocatalyzers tomodify glucans (maltooligosaccharides and starch) with GlcN building blocks bymeans of cell free glycobiology procedures.