STUDIES ON THE MECHANISM OF BACTERIAL BIOLUMINESCENCE IN VIVO AND IN VITRO

Persistent Link:
http://hdl.handle.net/10150/195376
Title:
STUDIES ON THE MECHANISM OF BACTERIAL BIOLUMINESCENCE IN VIVO AND IN VITRO
Author:
Campbell, Zachary Taylor
Issue Date:
2009
Publisher:
The University of Arizona.
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.
Abstract:
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.
Type:
text; Electronic Dissertation
Keywords:
Bioluminescence; Fre; Frp; Luciferase; Lux; Oxidoreductase
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Biochemistry & Molecular Biophysics; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Baldwin, Thomas O.
Committee Chair:
Baldwin, Thomas O.

Full metadata record

DC FieldValue Language
dc.language.isoENen_US
dc.titleSTUDIES ON THE MECHANISM OF BACTERIAL BIOLUMINESCENCE IN VIVO AND IN VITROen_US
dc.creatorCampbell, Zachary Tayloren_US
dc.contributor.authorCampbell, Zachary Tayloren_US
dc.date.issued2009en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © 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.en_US
dc.description.abstractDespite 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.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectBioluminescenceen_US
dc.subjectFreen_US
dc.subjectFrpen_US
dc.subjectLuciferaseen_US
dc.subjectLuxen_US
dc.subjectOxidoreductaseen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineBiochemistry & Molecular Biophysicsen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorBaldwin, Thomas O.en_US
dc.contributor.chairBaldwin, Thomas O.en_US
dc.contributor.committeememberCordes, Matthew J.en_US
dc.contributor.committeememberMcEvoy, Megan M.en_US
dc.contributor.committeememberMiyashita, Osamuen_US
dc.contributor.committeememberTama, Florenceen_US
dc.contributor.committeememberWright, Stephen H.en_US
dc.identifier.proquest10469en_US
dc.identifier.oclc659752090en_US
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