Investigation of Iron-Sulfur Cluster Involvement in Catalysis by the Hydro-lyase Quinolinate Synthase
Open Access
- Author:
- Griffiths, Amy E.
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- None
- Committee Members:
- Squire J Booker, Thesis Advisor/Co-Advisor
- Keywords:
- iron-sulfur
NadA
quinolinate synthase - Abstract:
- Quinolinate synthase catalyzes the formation of quinolinic acid, a key intermediate in the biosynthesis of the cofactor nicotinamide adenine dinucleotide. In prokaryotes, quinolinic acid is synthesized via the concerted action of two enzymes, L-aspartate oxidase, and quinolinate synthase (NadA). L-aspartate oxidase, a flavin-dependent enzyme that converts L-aspartic acid to iminoaspartate, has been well-characterized. In contrast, few studies have been conducted on NadA, which catalyzes a condensation reaction between iminoaspartate and dihydroxyacetone phosphate to form quinolinic acid. Recent work has found that Escherichia coli NadA contains a [4Fe-4S]2+, and that this cluster may serve a direct role in catalysis. Further support for the role of the iron-sulfur cluster in catalysis is established here through investigations of NadA from E. coli, Mycobacterium tuberculosis, and Pyrococcus horikoshii. While initial work with E. coli NadA has established the presence of an iron-sulfur cluster, this work indicates its necessity for catalysis. Isolation of a cluster-less apoprotein is described, and subsequent spectroscopic and activity analysis of the apoprotein indicates that is in fact unable to catalyze the formation of quinolinic acid. Additionally, the cluster can be chemically reconstituted in this form of the protein, yielding a catalytically active form of NadA, with characteristics nearly identical to the holoprotein previously described. An understanding of NadA from M. tuberculosis could be of use in designing new antimicrobial drugs to combat this deadly human pathogen. Although the M. tuberculosis NadA sequence does not contain the CX2CX2C motif that is commonly found in 4Fe-4S enzymes it does possess an iron-sulfur cluster similar to that in E. coli. A recent report of the crystal structure of NadA from the archeon P. horikoshii describes catalytic activity of the protein in the absence of an iron-sulfur cluster. To reproduce this observation independently, the P. horikoshii nadA gene was cloned and the protein purified under anaerobic conditions. The isolated protein did in fact contain a [4Fe-4S]2+ cluster in its catalytically active form, while apoprotein, or protein isolated under aerobic conditions showed no activity. The lack of cluster in the previously reported structure is believed to result from oxygen exposure. To gain further understanding of this enzyme and the role of its cluster in catalysis, studies were initiated to obtain a crystal structure of the protein with an intact cluster.