Substrate Positioning and Channeling Of Escherichia Coli Quinolinate Synthase
Open Access
- Author:
- Sites, Lauren Anne
- Graduate Program:
- Chemistry
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- October 19, 2012
- Committee Members:
- Squire J Booker, Thesis Advisor/Co-Advisor
- Keywords:
- Quinolinate Synthase
NadA
iron-sulfur cluster
NAD biosynthesis - Abstract:
- The essential cofactor nicotinamide adenine dinucleotide (NAD) is consumed in many metabolic reactions in the cell, necessitating the need to synthesize NAD. In most bacteria, the de novo pathway to form NAD begins with two unique enzymes that have been extensively studied herein. The first enzyme in the pathway, L-aspartate oxidase, performs a two-electron oxidation of L-aspartate to form iminoaspartate. This flavin containing enzyme can undergo multiple catalytic turnovers given the oxidants, fumarate or molecular oxygen, to afford the oxidized form of the enzyme. The second enzyme in the pathway, quinolinate synthase or NadA, condenses iminoaspartate and dihydroxyacetone phosphate to form quinolinic acid, the backbone of the pyridine ring of NAD. Many have postulated that these two enzymes can operate as an enzyme complex, yet no substantial evidence of this complex has been demonstrated. Investigations to examine the possible protein-protein interactions of the two enzymes were carried out, yet no obvious interaction was seen by the techniques employed. The oxygen-sensitive nature of Fe/S cluster of NadA and the kinetics of NadB prevented gathering data to conclude a concrete finding. The mechanism of NadA is still unknown despite discovering its requirement for a [4Fe-4S] cluster for activity over seven years ago. While it was speculated and now shown by others, that the Fe/S cluster acts a Lewis acid to aid in the final dehydration of a reaction intermediate to form quinolinic acid, no evidence exists with native substrates coordinating to the Fe/S cluster. The advanced spectroscopic techniques, EPR and ENDOR, were employed to examine NadA with various substrates. While it is known what carbon of DHAP is closest to the Fe/S cluster, an ENDOR signal was not seen when NadA was incubated with 13C-labeled DHAP.