Despite the importance of molecular recognition in nearly all aspects of protein function, the determinants of specificity for enzyme-substrate and protein-protein interactions are poorly understood. The majority of these complexes involving bacterial luciferase from V. harveyi have yet to be fully characterized. Luciferase catalyzes the reaction of molecular oxygen, FMNH2 and a long-chain aliphatic aldehyde yielding FMN, the corresponding carboxylic acid and blue-green light. In vivo, luciferase is thought to obtain FMNH2 following transfer from a transiently associated oxidoreductase. To identify the oxidoreductase responsible for providing FMNH2 in E. coli, bioluminescence was compared using single gene deletion strains deficient in either a homolog to the endogenous V. harveyi oxidoreductase (Frp) or an oxidoreductase distantly related to luxG from V. fischeri (Fre). Fre is responsible for reducing flavin in vivo but does not physically interact with luciferase. The association between luciferase and the flavin product is also described. Luciferase was crystallized and subjected to soaking with high concentrations of FMN. A model was obtained for luciferase bound to FMN. Using molecular dynamics, models for the enzyme:aldehyde, enzyme:FMNH2, and luciferase bound to several reaction intermediates are presented. Finally, a conserved loop region adjacent to the active center was investigated for the ability to facilitate protein:protein interaction between luciferase and the endogenous Frp oxidoreductase. Following alanine mutagenesis of the charged residues throughout this loop, it appears that the residues targeted by this study are not components of a docking platform but facilitate a lid-gating mechanism of paramount importance for catalytic function.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/195376 |
| Date | January 2009 |
| Creators | Campbell, Zachary Taylor |
| Contributors | Baldwin, Thomas O., Baldwin, Thomas O., Cordes, Matthew J., McEvoy, Megan M., Miyashita, Osamu, Tama, Florence, Wright, Stephen H. |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | English |
| Detected Language | English |
| Type | text, Electronic Dissertation |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
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