Publication Date

8-2014

Date of Final Oral Examination (Defense)

6-27-2014

Type of Culminating Activity

Thesis

Degree Title

Master of Science in Chemistry

Department

Chemistry

Supervisory Committee Chair

Rajesh Nagarajan, Ph.D.

Supervisory Committee Member

Don Warner, Ph.D.

Supervisory Committee Member

Julia Oxford, Ph.D.

Abstract

Gram-negative bacteria use acyl-homoserine lactone (AHL) based quorum sensing (QS) to regulate the expression of genes that give the bacteria a selective advantage over host defenses and antibiotic treatment. Burkholderia mallei is an antibiotic resistant pathogen that causes Glanders disease. B. mallei BmaI1 AHL-synthase uses octanoyl-Acyl Carrier Protein (C8ACP) and S-adenosyl-L-methionine (SAM) to synthesize the AHL, octanoyl-homoserine lactone (C8HSL). Inhibiting AHL-synthases has been difficult because mechanistic and substrate specificity details for these enzymes are not well understood. Our goal was to determine how BmaI1 activity and enzymatic mechanism changes with nonspecific, variable acyl chain acyl-ACP substrates. We found that catalytic efficiency of nonspecific acyl-ACP substrates are drastically low compared to the native C8ACP substrate, in-line with tight signal specificity observed in vivo. In addition, substrates with lower catalytic efficiency also showed kinetic cooperativity while reacting with BmaI1. Our results suggest that substrates add by a preferred order, random sequential mechanism to BmaI1. Alternatively, BmaI1 could exist in two forms, where nonspecific substrates bind to less active enzyme form that leads to formation of an unproductive E.acyl-ACP complex. Apparently, only the native acyl-ACP substrate forms both a stable and productive E.acyl-ACP complex thus providing a molecular basis for substrate discrimination in QS signal synthesis in B. mallei.

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