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dc.creatorJones, Christine Michelle
dc.date.accessioned2015-09-03T15:24:57Z
dc.date.available2015-09-03T15:24:57Z
dc.date.created2013-05
dc.date.issued2013-02-04
dc.date.submittedMay 2013
dc.identifier.urihttp://hdl.handle.net/1969.1/154889
dc.description.abstractIn addition to their primary biological function, many proteins are at least moderately capable of catalyzing secondary, promiscuous activities that may have a major role in enzyme evolution. Mounting evidence supports the idea that new enzymes can evolve when natural selection optimizes these weak promiscuous activities. It is not known what features of promiscuous enzymes enable the evolution of new activities. The o-succinylbenzoate synthase (OSBS) family of proteins has a subfamily capable of conducting N-succinylamino acid racemase (NSAR) activity in vitro. Analysis of genomic operon context has indicated that many of these NSAR/OSBS proteins conduct the NSAR activity in vivo. A specific asparagine residue in the active site is conserved only in the NSAR subfamily and not the rest of the OSBS family and is suspected to function directly in the NSAR reaction. The residue was replaced in a member of the NSAR subfamily by the corresponding residue from a non-promiscuous OSBS via site-directed mutagenesis. If the asparagine plays a direct role in the chemistry of the racemization, there should be a significantly larger effect on the NSAR activity relative to OSBS activity. Preliminary results, however, indicate that the effect on OSBS activity is much larger than anticipated. We were also interested in the extreme sequence divergence within the OSBS family as a whole. Generally, homologous enzymes that catalyze the same reaction share at least 40% sequence identity. Sequence identity within the OSBS family is as low as 15%. We hypothesize that while OSBS activity was conserved, functionally important residues diverged within OSBS subfamilies. From the crystal structure of the OSBS from Thermosynechococcus elongatus, a member of the Cyanobacteria 1 subfamily, residues were selected for site-directed mutagenesis based on proximity to the active site and ability to orient ligand for binding and catalysis. Kinetics data from mutants indicates that these non-catalytic residues have significant impact on the efficiency of enzyme activity.
dc.format.mimetypeapplication/pdf
dc.subjectenzyme evolution
dc.subjectcatalytic promiscuity
dc.subjectactivity
dc.subjectpromiscuity
dc.subjectprotein function
dc.subjectprotein proimscuity
dc.subjectevolution
dc.subjectenzyme
dc.subjectOSBS
dc.subjectNSAR
dc.subjectenolase superfamily
dc.subjectenolase
dc.subjectbacteria
dc.subjectvitamin K
dc.subjectmenaquinone
dc.subject
dc.titleEvolution of Enzyme Specificity in the OSBS Family
dc.typeThesis
thesis.degree.departmentBiochemistry/Biophysics
thesis.degree.disciplineGenetics
thesis.degree.grantorHonors and Undergraduate Research
dc.contributor.committeeMemberGlasner, Margaret E
dc.type.materialtext
dc.date.updated2015-09-03T15:24:57Z


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