Study of duplicated galU genes in Rhodococcus jostii and a putative new metabolic node for glucosamine-1P in rhodococci

CEREIJO AE; KUHN ML; HERNÁNDEZ MA; BALLICORA MA; IGLESIAS AA; ALVAREZ HM; ASCENCIÓN DIEZ MD

BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2021

0304-4165

BackgroundStudying enzymes that determine glucose-1P fate in carbohydrate metabolism is important to better understand microorganisms as biotechnological tools. One example ripe for discovery is the UDP-glucose pyrophosphorylase enzyme from spp. In the genome, this gene is duplicated, whereas contains only one copy.MethodsWe report the molecular cloning of galU genes from and to produce recombinant proteins RjoGalU1, RjoGalU2, and RfaGalU. Substrate saturation curves were conducted, kinetic parameters were obtained and the catalytic efficiency (kcat/Km) was used to analyze enzyme promiscuity. We also investigated the response of GlmU pyrophosphorylase activity with different sugar-1Ps, which may compete for substrates with RjoGalU2.ResultsAll enzymes were active as pyrophosphorylases and exhibited substrate promiscuity toward sugar-1Ps. Remarkably, RjoGalU2 exhibited one order of magnitude higher activity with glucosamine-1P than glucose-1P, the canonical substrate. Glucosamine-1P activity was also significant in RfaGalU. The efficient use of the phospho-amino-sugar suggests the feasibility of the reaction to occur . Also, RjoGalU2 and RfaGalU represent enzymatic tools for the production of (amino)glucosyl precursors for the putative synthesis of novel molecules.ConclusionsResults support the hypothesis that partitioning of glucosamine-1P includes an uncharacterized metabolic node in spp., which could be important for producing diverse alternatives for carbohydrate metabolism in biotechnological applications.General significanceResults presented here provide a model to study evolutionary enzyme promiscuity, which could be used as a tool to expand an organism´s metabolic repertoire by incorporating non-canonical substrates into novel metabolic pathways.