Theses and Dissertations

Issuing Body

Mississippi State University

Advisor

Emerson, Joseph P.

Committee Member

Fitzkee, Nicholas C.

Committee Member

Mlsna, Todd E.

Date of Degree

12-10-2021

Document Type

Graduate Thesis - Open Access

Major

Chemistry

Degree Name

Master of Science (M.S.)

College

College of Arts and Sciences

Department

Department of Chemistry

Abstract

Some bacteria produce a potent antifungal agent (pyrrolnitrin) from tryptophan using four dioxygen dependent steps to outcompete other microbes. Each step of this process 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 characterized from P. fluorescens. The pyrrolnitrin biosynthesis pathway (prnABCD) has been also observed in the Burkholderia genus. This thesis comprises my studies on the expression, purification, and characterization of PrnA from Burkholderia ambifaria. Beyond the comparative preliminary data on B. ambifaria 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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