Chemistry and Chemical Biology ETDs

Publication Date

8-28-2012

Abstract

My doctoral research primarily focuses on the Escherichia coli hotdog-fold thioesterases ydiI and ybgC. The ydiI gene is colocalized in the operon ydiHIJ. A substrate screen provided evidence that YdiI prefers aryl-CoA substrates and discriminated against the analogous aryl-ACP, unlike the paralog YbdB which works on 2,4-DHB-EntB in the enterobactin synthesis pathway. A bioinformatic approach showed that in some bacteria, ydiI is colocalized with genes from the menaquinone pathway. YdiI was shown to catalyze the hydrolysis of DHNA-CoA with physiological relevance (kcat/KM ~ 105). Furthermore, the E. coli YdiI- strain was shown to have perturbed growth, in good agreement with other menaquinone enzyme knockout experiments. Taking into account the evidence provided, YdiI is likely the DHNA-CoA thioesterase in the menaquinone pathway. Within the substrate binding pocket, changes to the YdiI Val68 to the YbdB equivalent of Met resulted in perturbed catalytic efficiency towards lauroyl-CoA, however not towards its physiological substrate DHNA-CoA. The YbdB M68V mutant resulted in increased efficiency towards lauroyl-CoA and DHNA-CoA, seemingly making it a gatekeeper. Secondly, YdiI catalyzes the hydrolysis of acyl-CoA's using a Glu63, His54, and Gln48 triad determined by mutagenesis experiments. The utilization of 18-O incorporation and rapid-quench techniques provided insight into YdiI's catalytic mechanism. A single phase multiple turnover reaction and the incorporation of 18-O in single turnover reactions suggests that ydiI uses a general base catalytic mechanism where Glu63 is the activating residue. The second E. coli hotdog-fold thioesterase protein discussed within is YbgC. YbgC is encoded by the first ORF of the tol-pal gene cluster. Previous work on the H. influenzae homologue demonstrated affinity for short chain acyl-CoA's and tandem affintity purificiation experiments showed E. coli YbgC co-purifies with ACP. No previous attempts to screen for acyl-ACP have been made. To screen YbgC for ACP activity, a method was developed to stoichiometrically construct acyl-ACP's, using apoACP, BF1558 acyl-transferase and acyl-CoA. The E. coli YbgC demonstrated preference for long chain acyl-CoA's and their analogous acyl-ACPs. To understand the nature of ACP binding to YbgC, SAXS analysis was carried out on the complex demonstrating that 2 ACP molecules bind to 1 dimer of YbgC.

Language

English

Keywords

hotdog-fold, thioesterase, YdiI, YbgC, acyl-ACP, menaquinone, coenzyme a

Document Type

Dissertation

Degree Name

Chemistry

Level of Degree

Doctoral

Department Name

Department of Chemistry and Chemical Biology

First Committee Member (Chair)

Mariano, Patrick

Second Committee Member

Bear, David

Third Committee Member

Melancon, Charles

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