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

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
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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.