Publication Date

5-2015

Advisor(s)

Erika Taylor

Department

Chemistry

Language

English

Abstract

Heptosyltransferase I (HepI), an enzyme of the glycosyltransferase B (GT-B) superfamily, catalyzes the addition of the first heptose (Hep) moiety of lipopolysaccharides (LPS) from the donor substrate, ADP-L-glycero-D-manno-Heptose (ADP-Heptose), to the acceptor substrate, KDO2-lipid A, through a inversion of stereochemistry about the anomeric carbon. HepI was previously structurally characterized, but these structures revealed little about acceptor substrate specificity or mechanism of catalysis. Escherichia coli HepI kinetics have been determined for KDO2-lipid A and KDO2-lipid A analogues demonstrating greater catalytic efficiency for O-deacylated and fully deacylated KDO2-lipid A (ODLA and FDLA) analogues (1.2 X 106 M-1 s-1 and 2.7 X 106 M-1 s-1, respectively) than native substrate KDO2-lipid A, 2.2 X 105 M-1 s-1. Additionally, these kinetics demonstrating that HepI is the first enzyme in the synthesis of LPS that does not have a dependence on the acylation state of its substrate. Changes in HepI intrinsic fluorescence, which are dependent on ODLA binding, suggest conformational changes occurring upon interacting with the acceptor substrate. Furthermore, changes in HepI rate with increasing concentrations of microviscogens indicating that conformational changes are partially rate limiting. Transient kinetics obtained by titrating HepI with ODLA demonstrate initial substrate concentration dependence and then become concentration independent, suggest a two step binding mechanism for ODLA. From these experiments the KD for ODLA was determined to be ~3 ΞΌM and a dynamic motion that occurs at a rate of ~74 s-1 was observed. Thermal melts of the protein indicate an increase in HepI stability in the presence of ODLA when the folding is monitored by circular dichroism. KIE experiments were attempted, however, due to unforeseen issues with radioactivity quenching, the data acquired is too preliminary to be conclusive. Several HepI homologues with varying degrees of sequence similarity to E. coli HepI were selected to evaluate structural differences required for binding their acceptor substrates, and significant advances have been made toward determining protein expression conditions for this library of enzymes. Of the homologues that have been cloned, conditions for large scale protein expression have been found for the Salmonella enterica HepI homologue. Through the combined investigations of HepI's catalytic reaction and structural dynamics it is hoped to design a tight binging inhibitor.

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