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|Title:||Discovery of Function in the Enolase Superfamily|
|Doctoral Committee Chair(s):||Gerlt, John A.|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||In the genomic era, advanced sequencing techniques have enabled an exponential growth of the protein sequence databases. Although the abundance of genomic sequences is valuable, the correctness of the gene annotations poses a problem--approximately one-half of the deposited sequences are incorrectly annotated. We are using the enolase superfamily as a model system to try to solve that problem. To date, >4000 members have been identified in the enolase superfamily, of which ∼50% have unknown functions. The muconate lactonizing enzyme (MLE) subgroup contains a family of enzymes that catalyze the epimerization of dipeptide substrates. Identification of function of unknown members of the MLE subgroup, based on sequence information and homology modeling, has been successful. A novel D-Ala-D/L-Ala and a unique L-Ala-D/L-Glu epimerase (AEE) were identified and kinetically characterized from Cytophaga hutchinsonii (k cat/KM of 5.5 x 104 M -1s-1) and Bacteroides thetaiotaomicron (kcat/KM of 5.8 x 104 M-1s-1), respectively. Computational predictions, provided by Prof. Matthew Jacobson's laboratory at UCSF, were proven correct for both enzymes. In collaboration with Prof. Steven Almo's laboratory at Albert Einstein College of Medicine, an X-ray crystal structure was solved at 1.6 A resolution for the B. thetaiotaomicron AEE, establishing the correctness of the predicted homology model.
Many enzymes in the mandelate racemase (MR) subgroup catalyze dehydration of various acid-sugar substrates. Attempts were made to elucidate the biochemical function of previously uncharacterized MR subgroup members from Agrobacterium tumefaciens, Mesorhizobium loti, Polaromonas sp., Silicibacter sp. and Sinorhizobium meliloti.
The glucarate degradation operon in Escherichia coli encodes two enolase superfamily members: D-glucarate dehydratase (GlucD) and its catalytically impaired close homologue, GlucD related protein (GlucDRP). However, the function of GlucDRP is unclear. New biochemical evidence suggests that GlucD and GlucDRP are interaction partners in vivo. The role of several residues in catalysis and substrate recognition from the capping domain loops were investigated via a mutagenesis study of GlucD.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009.
|Date Available in IDEALS:||2014-12-17|