Study On Subunits Interaction Of Acetohydroxyacid Synthase

China is a large agricultural country with large population and little arable land, hence the growth and sustainable development of agriculture is the foundation of economic prosperity and social stability. The pesticide has greatly improved agricultural production, but also faces the problem of pesticide resistance, duing to overuse or/and single mechanism for long-term use. How to use the proved ultra-efficient target enzymes to develop new green pesticides and solve the resistance problem has became a global scientific issue.Acetohydroxyacid synthase (AHAS, EC2.2.1.6), which is an important target of action of several commercialized herbicide with high herbicidal efficience, catalyzes the first common step in the biosynthesis of branched chain amino acids (valine, leucine and isoleucine). AHAS is composed of catalytic and regulatory subunits and the enzyme exhibits full activity only when the regulatory subunit (RSU) binds to the catalytic subunit (CSU). Previous results have suggested that the losing of the RSU has the considerable effect on AHAS activation with the herbicides inhibition. Addtionally, the heterologous activation among subunits of AHAS impled that there was a common interface between the CSU and the RSU. Hence, the common interface of AHAS is proposed to be a potential target for developing the new inhibitor with new action mechanism.The crystal structure of the holoenzyme has not been reported yet, and the molecular interaction between the CSU and the RSU is also unknown. The mechanism of catalytion, activation and feedback inhition was poorly understood. Therefore, the sduty on subunits interaction of AHAS is very important to develop the new pesticide. Work in this thesis was carried out as following:1. The global-surface, site-directed labeling scanning method is established to efficiently identify the key regions and residues for protein-protein interactions. Combined with the site-directed mutagenesis and computational approach, a protocol was established for the identification of the molecular interactions between proteins.2. Based on the protocol, the subunits interaction of E. coli AHAS III was studied. The Arg26and Asp69of the regulatory subunit were identified to be the key residues to interact with the catalytic subunit. The results of enzyme activation and pulldown experiment both showed that Arg26and Asp69were the key residues for subunits interaction with cooperative action and the charge of side-chain of Arg26and Asp69played an important role on the subunits interaction. A plausible protein-protein interaction model of the holoenzyme of E. coli AHAS III was proposed, based on the mutagenesis and protein docking studies.3. The mutant ilvl’, which was constructed by substituting all cyscines in the catalytic subunit of E. coli AHAS III (ilvl) with alaines, showed comparable activity with the wild-type ilvl. Hence the ilvl’ was able to instead of ilvl in the expriment. The potential interaction region of ilvl was determined to be around the residue Ter308using global-surface, site-directed labeling scanning method. The mutation in the dimer interface of ilvl showed that:the aggregation state of ilvl had effect on the enzymatic activity and interaction with ilvH. Further experimental data on the thermodynamics of subunit associations will be required for further understanding.4. The nature of reaction buffer has a significant impact on subunit interaction of AHAS. The results of pH dependency experiment showed that the polarity of the environment was conducive to subunits interaction, suggesting that there were more polar residues at the interface. AHAS catalytic reaction requires a divalent metal ion as a coenzyme but it does not have high specificity. The activity of E. coli AHAS Ⅲ can be activated by Mg2+, Ca2+, Sr2+and Ba2+. The enzymatic activity of both the isolated CSV and the reconstituted holoenzyme decreased with increasing the metal ion radius, but the activation ability of the RSU to the CSU increased.5. The minimum peptide of ilvH to activate ilvl. was determined to be the ΔN14-ΔC89, which comprises a full ACT domain. ΔN14-ΔC89was insensitive to valine inhibition, suggesting different elements for enzymatic activation and feedback regulation. The results of enzymatic activition and microscale thermophoresis (MST) showed that this peptide could not only activate and bind to its homologous ilvI and heterologous ilvB (CSU of E. coli AHAS I), but also heterologously activate and bind to the CSUs of AHAS from Saccharomyces cerevisiae, Arabidopsis thaliana and Nicotiana plumbaginifolia. The high sequence similarity of the peptide ΔN14-ΔC89to RSUs across species hints that this peptide represents the minimum activation motif in RSU and that it regulates all AHASs. These results would shed light on the design of inhibitor or regulator molecular targeted for PPI of AHAS.6. The RSU of AHAS contains the typical ACT domain, like all the other ACT domains, binding with the effector (BCAA, branched chain amino acids) to regulate the enzymatic activity. The inhibition efficiency of the BCAA was about80%, considerable with sulfonylurea and imidazolinone herbicides. The inhibitory activity of the20encoding amino acids was screened and the BCAA was the most efficient. The inhibition efficiency of the BCAA was considerable with sulfonylurea and imidazolinone herbicides. Therefore, to mimic the feedback regulation of the BCAA could develop the new pesticide active lead compounds with a novel action mechanism. The ACT domain is an evolutionarily mobile ligand binding regulatory module that has been fused to different enzymes at various times. The average sequence similarity of the ACT domian is only16%, but the three-dimensional structure is highly similar (RMSD-2.5A). The AHAS minimum activation peptides comprises a full ACT domain with cross-species activate ability, which triggered the thinking of the existence of the potential and broad spectrum regulatory networks of the ACT domain. The regulatory subunit of E. coli AHAS II (ilvM) has low sequence similarity in the RSU of AHAS (13-22%), but the ilvM can increase the activity of the CSU from E. coli, S. cerevisiae, A. thaliana and N. plumbaginifolia by8-16times. These results confirmed our idea of regulatory networks of the ACT domain. Three ACT domain proteins of different passways had been designed to activate the CSU of AHAS. The activation results suggested that the enzymatic activity of the CSU increased1-2times with the ACT proteins, verifing the presence of a potential and broad spectrum regulatory networks of the ACT domain in different passways.In summary, this thesis first established a global-surface, site-directed labeling scanning method to rapidly and efficiently identify the key interaction regions and residues for PPI. Combined with the site-directed mutagenesis and computational approach, a protocol was established for determining the molecular interactions of the PPI. Using this protocol, the key residues for subunits interaction of E. coli AHAS III have been indentified and a plausible PPI model of the holoenzyme of E. coli AHAS Ⅲ is proposed. The activation and regulatory mechanism of AHAS has been preliminary studied. The effect of reaction buffer on subunit interaction of AHAS has been discussed. The minimum activation peptide has been determined, which comprises a full ACT domain. This peptide could activate catalytic subunits across the species and has a high sequence similarity with RSUs, suggesting that it represents the minimum activation motif in RSU and regulates all AHASs. The inhibition efficiency of the BCAA was considerable with sulfonylurea and imidazolinone herbicides. Therefore, to mimic the feedback regulation of the BCAA could develop the new pesticide active lead compounds with a novel action mechanism. The ACT domain is an evolutionarily mobile ligand binding regulatory module that has been fused to different enzymes at various times. The AHAS minimum activation peptides comprises a full ACT domain with cross-species activate ability, which triggered the thinking of the existence of the potential and broad spectrum regulatory networks of the ACT domain in different passways.