INVESTIGATING STRUCTURE AND SUBSTRATE-BINDING OF A NOVEL GLUCO-TYPE KINASE NAHK

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Uridine 5?-diphospho -N-acetylglucosamine (UDP-GlcNAc) is an essential donor for synthesis of a diverse array of glycans & glycoconjugates used in fields spanning from therapeutic medicine and vaccines to food industry. In nature, various organisms employ different and mostly complicated pathways to synthesize UDP-GlcNAc for biosynthesis of cell walls, glycosaminoglycans such as hyaluronic acid, etc. The chemical synthesis of UDP-GlcNAc is also complicated and costly. The final goal of our project is to engineer a simple, economic and efficient enzymatic synthesis of UDP-GlcNAc. In order to accomplish this we are using a two enzyme system with GlcNAc and ATP as starting material. GlcNAc is converted to GlcNAc-1-P by an enzyme called NahK while utilizing ATP. GlcNAc-1-P is further converted to UDP-GlcNAc by GlmU while utilizing UTP. Whereas GlmU has been characterized structurally and biochemically, NahK is not. The specific focus of current project is to investigate the structure of NahK and study its binding with various substrates and substrate analogs. It is hoped that these studies will eventually lead to development of a more stable and efficient enzyme with optimum substrate specificity. NahK (B.longum) is a soluble acidic enzyme of moderate molecular weight (~ 40 kD). The protein belongs to protein kinase superfamily & functions as N-acetylhexosamine kinase. It is a relatively novel and unique enzyme in being the first reported enzyme that acts as a gluco-type kinase (ref. #1) i.e., can phosphorylate glucose and its analogs. Crystallization attempts were undertaken to obtain structure of this enzyme. Small crystals of 40-50 microns in largest dimension could finally be obtained. Being too small they could diffract only poorly at the home X-ray source (5 angstrom resolution). Selenomethionine containing protein was obtained by using methionine auxotrophic host strain. The crystals obtained from this Se-protein would be tried for MAD studies.