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Molecular Characterization and Expression of an Oxidase from Field Bean (Dolichos lablab)

Santosh, R. Kanade (2007) Molecular Characterization and Expression of an Oxidase from Field Bean (Dolichos lablab). PhD thesis, University of Mysore.

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Abstract

Polyphenol oxidases (PPO) are type III enzymes with a dinuclear copper centre which initiates enzymatic browning. PPOs are mixed function oxidases which catalyze both the hydroxylation of monophenols to diphenols (monophenolase/cresolase) and also oxidation of o-diphenols to o-quinones. The PPO has been purified to apparent homogeneity. The purified enzyme had a specific activity of 35553 U/mg with a yield of 20 %.The PPO was found to be a hetero dimer of 29000 Da and 31000 Da with a native molecular mass of 120000 Da. The amino-terminal sequence of the subunits are identical. The kinetic studies revealed that tertiary butyl catechol is the best substrate followed by 4-methyl catechol and catechol. The purified protein was a glycoprotein, the neutral sugar composition being 8.0 % and pI of 9.3. Both the subunits of PPO cross react with PPO antibodies. The spatial and temporal expression of PPO during germination and seed development were evaluated. A series of phenolic compounds experimentally evaluated for their binding affinity and inhibition constants were computationally docked to the active site of catechol oxidase. Analyses of the complexes provide structural explanations for correlating subtle changes in the position and nature of the substitutions on diphenols to their functional properties as substrates and inhibitors. Higher reaction rates and binding are reckoned by additional interactions of the substrates with key residues that line the hydrophobic cavity. The docking results suggest that inhibition of oxidation stems from an interaction between the aromatic carboxylic acid group and the apical His109 one of the four co-ordinates of the trigonal pyramidal co-ordination polyhedron of CuA. The spatial orientation of the hydroxyl in relation to the carboxylic group either allows a perfect fit in the substrate cavity leading to inhibition or due to a steric clash flips the molecule vertically facilitating oxidation. This is the first study, which explains at the molecular level the determinants of substrate/inhibitor specificity of a catechol oxidase. The enzyme is activated manifold either in the presence of the anionic detergent SDS below its critical micellar concentration or on exposure to acid pH. The activation is accompanied by a marked shift in the pH optimum enhanced kcat, an increased sensitivity to the competitive inhibitor tropolone, altered susceptibility to proteolytic degradation and decreased thermal 6 stability. The activation is characterized by a unique and large increase in the Stokes radius. The activation is due to a localized conformational change that is anchored around the active site. The BLAST search of internal peptide sequences indicated a high homology (>90%) to the galactose specific lectins of legumes. The cDNA sequence of 786 bp was obtained using degenerate primers corresponding to the amino-terminal sequence and internal peptide sequence and submitted to Gene Bank (Accession No.EF204527) This sequence is highly homologous to galactose specific legume lectins. Consequently the protein was purified by two independent methods and multifunctional property was characterized.

Item Type: Thesis (PhD)
Uncontrolled Keywords: Polyphenol oxidases (PPO) field bean enzymes molecular characterization
Subjects: 600 Technology > 08 Food technology > 22 Legumes-Pulses
500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 16 Enzyme Chemistry
Divisions: Protein Chemistry and Technology
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 07 May 2009 05:12
Last Modified: 28 Dec 2011 10:07
URI: http://ir.cftri.com/id/eprint/8926

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