Publication Date

5-2018

Date of Final Oral Examination (Defense)

10-12-2017

Type of Culminating Activity

Thesis

Degree Title

Master of Science in Chemistry

Department

Chemistry

Supervisory Committee Chair

Rajesh Nagarajan, Ph.D.

Supervisory Committee Member

Eric Brown, Ph.D.

Supervisory Committee Member

Owen McDougal, Ph.D.

Abstract

Quorum sensing is an intercellular mechanism used by many bacterial pathogens to regulate behaviors, such as motility, virulence, and antibiotic production. Disruption of quorum sensing is shown to be deleterious to bacterial pathogenicity, without causing lethality. In Gram-negative bacteria, acyl-homoserine lactone (AHL or acyl-HSL) signal molecules are synthesized by AHL synthase enzymes using acylated-acyl carrier protein (acyl-ACP) and S-adenosyl-L-methionine (SAM) as their substrates. Pantoea stewartii EsaI, which causes Stewart’s wilt in corn, and Yersinia pestis YspI, which causes bubonic plague are AHL synthases that use 3-oxohexanoyl-ACP and 3-oxooctanoyl-ACP as the acyl-substrate to synthesize 3-oxohexanoyl-HSL and 3-oxooctanoyl-HSL signals, respectively. Unfortunately, the instability of the β-ketoacyl-ACP substrate due to the enolate formation at the β-position in the acyl-chain impedes mechanistic investigations for β-ketoacyl-ACP utilizing enzymes. In this thesis, we designed, developed, and evaluated a library of stable β-ketoacyl-ACP mimics for two β-ketoacyl-ACP utilizing AHL synthases, EsaI and YspI. We found that 2-furanacetyl-ACP and 2-benzofuranacetyl- ACP were the best 3-oxoacyl-ACP alternative substrate for EsaI and YspI, respectively, and within an order of magnitude to that observed for well-characterized ACP/CoA- dependent AHL synthases with their native acyl-ACP/ acyl-CoA substrate, such as RhlI, BmaI, and BjaI. The presence of a heteroatom other than oxygen is crucial to retain enzyme activity in both EsaI and YspI. Also, substrate inhibition of 2-thiopheneacetyl-ACP was observed with both enzymes. The success of synthesis and high activity of β-ketoacyl-ACP mimics for EsaI and YspI should open new doors in characterizing this class of enzymes. Moreover, the β-ketoacyl-ACP substrates could be used as chemical probes to explore and design inhibitors for therapeutically important AHL synthases and several uncharacterized enzymes that impact human health, such as β-ketoacyl-ACP reductase in fatty acid biosynthesis and polyketide synthase in polyketide synthesis which are targets for antimicrobial, antimalarial, and anti-cancer drugs.

DOI

10.18122/td/1395/boisestate

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