| Summary: | 碩士 === 國立中興大學 === 生物化學研究所 === 96 === T2SS is the mechanism required for Xanthomonas campestris pv. campestris to secret hydrolytic enzymes in the invasion of its host. Widespread in pathogenic bacteria, the secretion system leads to animal and plant diseases. XpsE, a cytosolic protein, is the only component in T2SS that possesses ATP binding and hydrolyzing activities and may work as the molecular motor. Previous studies indicated that the N1 domain of XpsE is inhibitory to its ATPase activity exihibited by its C domain. An inner membrane protein XpsL acts as a positive regulator of the XpsE ATPase, presumably by binding to the XpsE N1 domain and alliviating its inhibition effect. An N2 domain physically located between N1 and C domain of XpsE may coordinate conformational change related ATPase activity regulation. XpsE forms homo-multimer and may function as hexamer in T2SS. Crystal structures of XpsE homologues suggest that the N2 domain may be involved in inter subunit association in hexamer. This study is focused on the analysis of an XpsE mutant that is mutated at the conserved residue D202 in the N2 domain. The XpsE(D202A) is no longer functional in secretion. It also interferes with normal secretion when introduced into the wildtype strain. In crystal structures of XpsE homologue, the D202 equivalent residues are close to ATP bound to the C domain. To get better understanding of functional role of the residue D202 in XpsE, ATP binding and ATP hydrolyzing activities of the mutant were analyzed in parellel with the wildtype XpsE. In most assays, no significant alteration was observed. The mutant remains capable of ATP binding, appears to exihibit normal oligomerization activity and XpsLN binding. However, it exihibits slightly lower intrinsic ATPase activity and deviated ADP binding. By inspecting surroundings of D202 equivalents in the solved crystal structures, we speculated that the D202 residue of XpsE may be required for steps downstream of ATP hydrolysis by XpsE.
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