The Structure And Function Of Human Gmp Reductase 2 (gmpr2)

Purine metabolism is essential in nucleoside metabolism, involving in the biochemical and physiological process, and plays key roles in cell growth, development, differentiation, proliferation, and cell apopotosis. Guanosine monophosphate reductase (GMPR, EC 1.7.1.7) catalyzes the irreversible and NADPH-dependent reductive deamination of GMP to IMP, and plays a critical role in reutilization of free intracellular bases and purine nucleosides and maintaining the balance of Adenosine and Guanosine in cell. However, it is unclear for GMPR about the reaction mechanism and functional research so far.In this study, GMPR2 fusion protein was expressed successfully in E.Coli cells and purified with 95% homogenicity. Complex of GMPR2 and substrate GMP was crystallized by using the method of hanging-drop vapor-diffusion at 4℃. X-ray diffraction analysis revealed that the crystals belong to the space group P3₂21 with unit-cell parameters a = b = 110.6 (?), c = 209.8 (?) (a=β=90°, γ =120°) , and they diffracted to at least 3.0 A resolution. The first crystal structure of GMPR family (Protein Data Bank entry 2A7R) was solved through the molecular replacement method using the 2.18 A resolution structure of Thermotoga Maritima IMP dehydrogenase as a search model (PDB entry 1VRD).The GMPR2 formed a tetramer composed of subunits adopting the ubiquitous (a/β)₈ barrel fold. Each subunit was comprised of 11a and 14β. Interestingly, the substrate GMP was bound to hGMPR2 through interactions with Met269, Ser270, Arg286, Ser288, and Gly290; this made the conformation of the adjacent flexible binding region (residues 268-289) fixed, much like door on a hinge. Structures comparison and sequence alignment analyses showed that the conformation of the active site loop (residues 179-187) was similar to those of hGMPR1 and Inosine Monophosphate Dehydrogenases (IMPDHs). We proposed that Cys186 was the potential active site, and that the conformation of the loop (residues 129-133) suggested a preference for the coenzyme NADPH over NADH. This structureprovides important information towards understanding the functions of members of the GMPR family.Site-directed mutations of several conserved amino acids were used to understand their characterizations according to the structural analysis of GMPR2. The results showed that Cysl86Ala and Thr188Ala abolished hardly the enzyme activity of GMPR2; Serl84Ala and Asnl32Ala caused partly inactivation related to the wild type of GMPR2. Reverse transcription-PCR analysis showed that GMPR2 was expressed ubiquitously in human tissues, liver adenocarcinoma, lung adenocarcinoma, and stomach adenocarcinoma tissues. Cluster analysis of microarray data revealed some GMPR-related genes, including APRIN and IFIT1, involved in cell proliferation. Subcellular location with green fluorescent protein (GFP) showed that GMPR2 was distributed in the cytoplama in COS-7 cells, while expressed both in cytoplama and nucleus in AD293 cells. The expression of GMPR2 was silenced by RNA interference (RNAi) method. The results showed it did little effection to the level of cell cycle (non-silencing RNA: 22.76±1.43%; within the three siRNA, GMPR2-1: 25.12±1.84 % ;GMPR2-2: 26.72±1.24 % ; GMPR2-3: 21.34±1.72 % ) and cell apopotosis. Overexpression of GMPR2 in AD293 cells resulted in the elevated level of cell proliferation (control: 37.12±1.28%; GMPR2: 58.36±1.53%), but no effect on cell apopotosis.The structural and functional researches of human GMPR2 were performed in this paper. We successfully resovled the first crystal structure of GMPR family (Protein Data Bank entry 2A7R) and analyzed its characterization and active site. Moreover, mutants, expressed profile in different tissues, subcellular location, and RNAi researches were used to study the function of human GMPR2.