title: Structural and mechanistic analysis of engineered trichodiene synthase enzymes from Trichoderma harzianum: towards higher catalytic activities empowering sustainable agriculture.
abstract: Trichoderma spp. are well-known bioagents for the plant growth promotion and pathogen suppression. The beneficial activities of the fungus Trichoderma spp. are attributed to their ability to produce and secrete certain secondary metabolites such as trichodermin that belongs to trichothecene family of molecules. The initial steps of trichodermin biosynthetic pathway in Trichoderma are similar to the trichothecenes from Fusarium sporotrichioides. Trichodiene synthase (TS) encoded by tri5 gene in Trichoderma catalyses the conversion of farnesyl pyrophosphate to trichodiene as reported earlier. In this study, we have carried out a comprehensive comparative sequence and structural analysis of the TS, which revealed the conserved residues involved in catalytic activity of the protein. In silico, modelled tertiary structure of TS protein showed stable structural behaviour during simulations. Two single-substitution mutants, i.e. D109E, D248Y and one double-substitution mutant (D109E and D248Y) of TS with potentially higher activities are screened out. The mutant proteins showed more stability than the wild type, an increased number of electrostatic interactions and better binding energies with the ligand, which further elucidates the amino acid residues involved in the reaction mechanism. These results will lead to devise strategies for higher TS activity to ultimately enhance the trichodermin production by Trichoderma spp. for its better exploitation in the sustainable agricultural practices.
authors.affiliation: a School of Earth and Environmental Sciences, Central University of Himachal Pradesh , District-Kangra, Shahpur , Himachal Pradesh 176206 , India.
authors.forename: Indu
authors.initials: I
authors.lastname: Kumari
authors.affiliation: a School of Earth and Environmental Sciences, Central University of Himachal Pradesh , District-Kangra, Shahpur , Himachal Pradesh 176206 , India.
authors.forename: Nitika
authors.initials: N
authors.lastname: Chaudhary
authors.affiliation: b School of Life Sciences, Central University of Himachal Pradesh , District-Kangra, Shahpur , Himachal Pradesh 176206 , India.
authors.forename: Padmani
authors.initials: P
authors.lastname: Sandhu
authors.affiliation: a School of Earth and Environmental Sciences, Central University of Himachal Pradesh , District-Kangra, Shahpur , Himachal Pradesh 176206 , India.
authors.forename: Mushtaq
authors.initials: M
authors.lastname: Ahmed
authors.affiliation: b School of Life Sciences, Central University of Himachal Pradesh , District-Kangra, Shahpur , Himachal Pradesh 176206 , India.
authors.forename: Yusuf
authors.initials: Y
authors.lastname: Akhter
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D019755:Carbon-Carbon Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D006860:Hydrogen Bonding; D008024:Ligands; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D055550:Protein Stability; D013329:Structure-Activity Relationship; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
chemical_list: D008024:Ligands; D019755:Carbon-Carbon Lyases; C051130:trichodiene synthetase
keywords: Trichoderma harzianum; biocontrol agent; catalytic site mutants; enzyme engineering; symbiosis; trichodiene synthase