Characterization Of The Biochemical Functions Of HetN And Study On FtsZ-HetN Protein-protein Interaction In Anabaena Sp. Strain PCC 7120

Anabaena sp. strain PCC 7120 is a filamentous diazotrophic cyanobacterium that is able to differentiate heterocysts in a semiregular pattern in response to combined nitrogen deprivation in the environment. Heterocysts provide a micro-oxic environment for the oxygen-sensitive nitrogenase complex, which catalyzes the reaction of nitrogen fixation. HetN, a putative ketoacyl reductase, is required for heterocyst pattern maintenance in the filamentous cyanobacterium Anabaena sp. strain PCC 7120. The hetN gene, when present on a mutli-copy plasmid, could suppress heterocyst differentiation. Little is known about the biochemical properties of HetN.In this study, we found that HetN could hydrolyze ATP or GTP in vitro, and this activity is dependent on the presence of magnesium in the reaction mixture. Mutations of the conserved active Ser142-Tyr155-Lys159 triad, predicted as necessary for the reductase activity of HetN, had only weak effect on the hydrolysis of ATP. The residue Lys159 is shown to be necessary for the heterocyst-suppressing activity of HetN, since the corresponding mutant allele present on a replicative plasmid failed to block heterocyst differentiation in contrast to the wild type. This result suggests that the reductase activity of HetN is involved in the HetN-mediated inhibition of heterocyst formation.In this study we also characterized the interaction of FtsZ and HetN by yeast two-hybrid system and pull-down assay. Overexpression of FtsZ in Anabaena sp. strain PCC7120 may form multiple contiguous heterocysts 48 hrs after combined nitrogen step-down, one mimic of the hetN insertional mutant. Interaction of FtsZ and HetN promoted the in vitro ATP hydrolysis activity by HetN in a linear relationship in accordance to the increase of FtsZ molar ratios in the reaction mixture. Effects of the mutations of the conserved active Ser142-Tyr155-Lys159 triad was also investigated in the interaction of FtsZ and HetN, Ser142 mutation is shown to enhance the interaction while Lys159 mutation is shown to lose the interaction. This result suggests that FtsZ-HetN interaction might be involved in the heterocysts pattern maintenance mechanism.This research about HetN biochemical properties and interaction of FtsZ and HetN opens a new angle for the study of the regulation network about heterocysts differentiation in Anabaena sp.strain PCC7120.