Initial characterization of PrnA from Burkholderia ambifaria: Developing an NADPH-dependent activity assay for Tryptophan halogenation - MS Defense

October 1, 2021

2:00 pm


Initial characterization of PrnA from Burkholderia ambifaria: Developing an NADPH-dependent activity assay for Tryptophan halogenation

Mahmuda Akter

Department of Chemistry
Mississippi State University

Friday, October 1, 2021
2:00 PM
Hand Lab 1100

Abstract


Pseudomonas species produce a potent antifungal agent (pyrrolnitrin) from tryptophan using four dioxygen dependent steps. Each step is catalyzed by an oxygenase encoded by the prnABCD cassette. The first enzymatic step in pyrrolnitrin biosynthesis is the regioselective chlorination of tryptophan to form 7-chlorotryptophan. This halogenation is catalyzed by PrnA, a flavin dependent oxygenase, which has been isolated and partially characterized from P. fluorescens. The pyrrolnitrin biosynthesis pathway (prnABCD) has been also observed in the Burkholderia genus. This thesis comprises the expression and purification of PrnA from Burkholderia ambiferia, which is associated with the biosynthetic pathway of the pyrrolnitrin. Here we also report our efforts to characterize Burkholderia ambifaria PrnA. Beyond the comparative preliminary data B. ambiaria PrnA, we report an NADPH-dependent activity assay for PrnA coupling the oxidative chlorination of tryptophan (and related substrates) with NADPH consumption. The steady-state kinetic parameters associated with PrnA of kcat, Km, and catalytic efficiency of enzymes are also reported for this system under defined experimental conditions.


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