title: A ripening associated peroxidase from papaya having a role in defense and lignification: heterologous expression and in-silico and in-vitro experimental validation.
abstract: Fruit ripening associated full length cDNA of a peroxidase from papaya was cloned and heterologously expressed. The expressed peroxidase was activated by in-vitro re-folding in the presence of hemin and calcium. The purified recombinant peroxidase exhibited broad substrate affinity in the order of o-dianisidine>pyrogallol>guaiacol and was found to be a homotetramer of 155kDa with each subunit having a size of 38kDa. The basis of the distinctive preferences for various substrates was investigated through in-silico molecular modeling approaches. Thus, when the modeled papaya peroxidase-heme complex was docked with these substrates, the in-silico binding efficiency was found to be in agreement with those of wet lab results with the involvement of Arg37, Phe40, His41, Pro137, Asn138, His139, His167, and Phe239 as the common interacting residues in all the cases. However, the binding of the different substrates were found to be associated with conformational changes in the peroxidase. Thus, in the case of o-dianisidine (the most efficient substrate), the protein was folded in the most compact fashion when compared to guaiacol (the least efficient substrate). Protein function annotation analyses revealed that the papaya peroxidase may have biological roles in oxidation-reduction processes, stresses, defense responses etc. In order to further validate its role in lignifications, the papaya peroxidase was compared with a lignin biosynthetic peroxidase from Leucaena leucocephala, a tree legume. Thus, based on 3D structure superimposition and docking, both peroxidases exhibited a great extent of similarity suggesting the papaya peroxidase having a role in lignification (defense response) too. The predicted functions of papaya peroxidase in defense response and lignification were further validated experimentally using qRT-PCR analyses and measurement of oxidation of coniferyl alcohol.
authors.affiliation: Department of Biochemistry, Bioinformatics Infrastructure Facility & Centre of Excellence in Bioinformatics, University of Lucknow, Lucknow 226007, U.P., India.
authors.forename: Veda P
authors.initials: VP
authors.lastname: Pandey
authors.affiliation: Department of Biochemistry, Bioinformatics Infrastructure Facility & Centre of Excellence in Bioinformatics, University of Lucknow, Lucknow 226007, U.P., India. Electronic address: upendradwivedi@hotmail.com.
authors.forename: Upendra N
authors.initials: UN
authors.lastname: Dwivedi
mesh_terms: D000595:Amino Acid Sequence; D029441:Carica / Q000201:enzymology* / Q000502:physiology; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D003962:Dianisidine / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D006139:Guaiacol / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000502:physiology*; D010940:Plant Proteins / Q000502:physiology*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011748:Pyrogallol / Q000737:chemistry; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D018076:DNA, Complementary; D010940:Plant Proteins; D011994:Recombinant Proteins; D011748:Pyrogallol; D006418:Heme; D006139:Guaiacol; D010544:Peroxidases; D003962:Dianisidine
keywords: Heterologous expression; In-vitro protein refolding; Molecular modeling and docking; Ni-NTA agarose affinity column chromatography; Rapid Amplification of cDNA Ends (RACE)