Cloning And Heterologous Expression Of OsHXK6 And The Catalytic Properties Analysis

Hexokinase (HXK; EC 1.1.1.14) has a number of isozymes functionally involved in the regulation of plant nutrient utilization including rice root ammonium assimilation and the related carbon catabolism. In this study, Oryza sativa L. cv indica 9311 and Oryza sativa L. cv Luoyou8 were used as the plant materials to investigate the role of OsHXKs in the α-ketoglutarate regulation of root ammonium assimilation.48 h treatment of 3 mM α-ketoglutarate or 90 mM sucrose increased GS and NADH-GOGAT activities in rice roots and ammonium uptake rate as well, which suggested the stimulatory effect of α-ketoglutarate on root ammonium assimilation. Activity staining analysis and qPCR experiments revealed that α-ketoglutarate treatment enhanced root OsHXK isoform activities and their mRNA transcript abundances, which suggested OsHXKs were the target proteins for α-ketoglutarate to modulate ammonium assimilation in rice roots.Total RNA was extracted from rice roots and subjected to reversed transcription for synthesis of the first chain cDNA, the template used for the PCR amplification of OsHXK6 full-length cDNA. The amplified fragment was inserted into pMD18-T for sequencing. Bioinformatics analysis indicated that the OsHXK6 was a membrane-associated hexokinase with a typical transmembrane region constituted by N-terminal 27 residues. Compared with the homolog isoform identified in Oryza sativa L cv japonica, the rice 9311 OsHXK6 exhibited five nucleotide acid mutations in the open reading frame that resulted in four amino acid substitutions in the hexokinase primary structure. The coding sequence of OsHXK6-27AA was PCR-amplified and inserted into expression vector pET28a to construct the recombinant expression plasmid for the E.coli Rosetta(DE3) transformation and the following IPTG-induced expression of the recombinant OsHXK6-27AA with the predicted molecular weight of 52.9 kDa. The optimum pH and temperature for the truncated hexokinase to phosphorylate glucose were 7.6 and 37℃, respectively. The Km of OsHXK6-27AA towards glucose and ATP were deterimined as 80.2 and 403.7μM, respectively. Meanwhile, the Hill coefficient was 1.3 for the substrate glucose and 1.0 for the ATP. The evidence that 40℃ incubation for one hour resulted in more than 80% loss of the enzyme activity suggested a relatively poor thermal stability for the recombinant OsHXK6-27AA. The investigated metal ions, including Fe(Ⅲ), Zn(Ⅱ) and Ca(Ⅱ) could acitivate OsHXK6-27AA activity towards glucose. On the contrary, Cu(Ⅱ) was an effective inhibitor for OsHXK6-27AA to phosphorylate glucose. The Km of recombinant OsHXK6-27AA expressed in P. pastoris GS115 strains was 62.7μM for glucose and 78.8μM for ATP, respectively. The Hill coefficient for the substrate glucose and ATP were 1.4 and 1.1, respectively. The reported hexokinase inhibitors, including 2-deoxy-D-glucose,3-O-methyl-glucose and N-acetyl-glucosamine, could effectively decrease OsHXK6-27AA activity towards both glucose and ATP in the present study. The ability order for the three competitive inhibitors were 2-deoxy-D-glucose, N-acetyl-glucosamine, and 3-O-methyl-glucose.In the present work, we reported that the OsHXKs were the target enzymes involved in the a-ketoglutarate regulation of rice root ammonium assimilation. Based on this observation, we further cloned and heterologously expressed the trucated protein of OsHXK6 in E.coli and yeast P. pastoris GS115. The important hexokinase properties, including substrate kinetics, inhibitor effects, and thermal stability, were studied for the recombinant OsHXK6-27AA. This research provided the necessary experimental evidences to further investigate the function of the hexokinase isozymes in regulating plant nitrogen assimilation.