Duplicated glgC genes in Kocuria rhizophila encode ADP-glucose pyrophosphorylases with different substrate specificity

IGLESIAS AA; IGLESIAS MJ; ASENCION DIEZ MD

Workshop; Primer Workshop virtual de la Red Argentina de Tecnología Enzimática; 2021

Red Argentina de Tecnologia Enzimatica (RedTEz)

Partitioning of glucose-1P (Glc-1P) is an important taskto comprehend carbohydrate metabolism in microorganisms and their potential asbiotechnological tools for glycan synthesis. In bacteria, UDP-glucose (UDP-Glc)and ADP–glucose (ADP-Glc) are built from glucose-1P by UDP-Glcpyrophosphorylase (EC 2.7.7.9) and ADP-Glc pyrophosphorylase (EC 2.7.7.27), dependingif the co-substrate is UTP or ATP, respectively. The former enzyme is encodedby galU genes while the latter by glgC genes. Kocuria rhizhophila is a relevant Actinobacteriabecause its ability to degrade phenolics compounds (such as those from lignin degradation)and tolerates 10% sodiumchloride in growth media. A genomic analysis shows that K. rhizophila presents a glgCgene duplication (glgC93 and glgC127) but a single galUcopy. Since gene duplications may lead to functional redundancies when the encodedproteins have the same activity or overlapping substrate ranges, criticalquestions arise given the putative scenario with two ADP-Glc pyrophosphorylases.The last-mentioned is the key regulated enzyme in microbial glycogen synthesis,allowing ADP-Glc synthesis under particular physiological circumstances.The twoKrhGlgC93 and KrhGlgC127 proteins  share 31% identity. Here, we present the characterization of KrhGlgC93,KrhGlgC127 and KrhGalU enzymes from K. rhizophila to understandcarbon split at the Glc-1P node. The molecular cloning, recombinant expressionand IMAC purification allowed the three enzymes to be obtained at a pureelectrophoretic degree. Kinetic analysis showed that KrhGalU is atypical prokaryotic UDP-Glc pyrophosphorylases and KrhGlgC127 presents similarproperties to other actinobacterial ADP-Glc pyrophosphorylases. Indeed, theenzyme showed a specific activity of 10.6 U/mg with S0.5values of 0.7 mM and 0.5 mM for ATP and Glc-1P, respectively. KrhGlgC127resulted to be an allosteric enzyme, highly activated by glucose-6P (A0.5lower than 0.4 mM), glucosamine-6P (A0.5: 0.86 mM) andpyruvate (highest activation at 2 mM).  Aspredicted, KrhGlgC93 was active with ATP and Glc-1P as substratesalthough exhibited substrate promiscuity toward NTPs (significatively activewith dTTP, CTP and GTP). Surprisingly, it depicted 5-fold higher activity whenUTP replaced ATP.Results presented support the hypothesis of analternative UTP-related enzyme acting in glucose-1P partitioning in K.rhizophila, opening questions regarding the fate of glucose moieties in theorganism. Further studies with KrhGlgC93 would be important foradvancing in the understanding of enzyme promiscuity from an evolutionary pointof view. In addition, this enzyme appears as an interesting candidate to beincorporated to cell-free glycobiology processes being developed in our groupinvolving NDP-sugar molecules.