The Protein Engineering of Peroxidases: The G45Y mutation of Ascorbate Peroxidase and Directed Evolution of Dehaloperoxidase

by Curme, Jonathan Woodward

Abstract (Summary)
Curme, Jonathan W B. Protein Engineering of Peroxidases: The G45Y mutation of ascorbate peroxidase and the directed evolution of dehaloperoxidase. (Under the direction of R. A. Ghiladi). The hemoprotein Ascorbate Peroxidase (APX) has an active site that is structurally similar to that of the bifunctional catalase-peroxidase (KatG). One notable difference between the two is the presence of a Gly residue in APX rather than the tyrosine that is normally found in KatG. The active site of KatG contains a modification of three amino acid residues, resulting in the formation of two covalent bonds which link the aforementioned tyrosine, Tyr229, to Trp107 and Met255 (Mycobacterium tuberculosis numbering). This Met-Tyr-Trp âcrosslinkâ has been suggested to impart catalase activity to the KatGs. To determine if the formation of such a crosslink and the catalase activity it may impart is unique to the KatGs, the APX mutant Gly45Tyr (Pisum sativum L. numbering) was expressed. While no increase in catalase activity was observed, this mutant did exhibit an increase in peroxidase activity: ~6-fold increase in kcat (G45Y, 2.43 s-1; WT, 0.40 s-1). A smaller change in Km was also noted (G45Y, 2.43*10-3; WT, 0.0011). Stopped-flow UV visible data indicates the presence of a new intermediate when G45Y is reacted with H2O2 when compared to WT APX, and we have attributed this change in reactivity to the tyrosine residue of the mutant. HPLC data suggests that the crosslink is not present, as does the lack of any catalase activity. The hemoprotein Dehaloperoxidase (DHP) is structurally similar to myoglobin but has peroxidase activity. DHP is found in Amphitrite ornata, a marine worm that coexists with other species that generate various polyhalogenated phenols to deter competition. DHP catalytically oxidizes halophenols to quinones, provided that there is a halogen para to the alcohol. This is of particular interest due to the fact that many polyhalogenated phenols are generated as chemical byproducts and waste from sewage treatment plants and pesticides. DHP mutants with higher substrate specificity for these byproducts could be used in bioremediation. We have epmployed directed evolution and epPCR to generate a library of mutants with enhanced dehaloperoxidase activity. Two screening techniques have been developed to identify mutants with enhanced peroxidase activity and enhanced selectivity for 2, 4-dihalophenol. The screening techniques have been tested using the wild type strain of DHP and the Tyr38Phe (Y38F) mutant of DHP. DHP(Y38F) possesses a higher turnover rate than the wild type. The peroxidase screening assay is colorimetric and DHP(Y38F) presents the oxidized form of the ortho-dianisadine substrate faster than the wild type enzyme. The presence of 2,4-dichlorophenol in the growth media inhibits the growth of E. coli and cells with DHP present will grow faster than those with out it. Comparison of LB agar plates grown with DHP(Y38F) and WT DHP containing cells have shown that the DHP(Y38F) grows larger colonies than the wild type, which indicates a higher substrate specificity of DHP(Y38F) for 2,4-dichlorophenol.
Bibliographical Information:

Advisor:Reza A. Ghiladi; Edmond F Bowden; Stefan Franzen

School:North Carolina State University

School Location:USA - North Carolina

Source Type:Master's Thesis



Date of Publication:08/19/2008

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