Discovery of function in the enolase superfamily

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 (kcat/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 Å 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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2010

Author dc:creator

• Lukk, Tiit

Contributors dc:contributor

• Gerlt, John A.
• Martinis, Susan A.
• Morrissey, James H.
• Nair, Satish K.

• enolase superfamily
• discovery of function
• dipeptide epimerase
• glucarate dehydratase
• acid sugar dehydratase
• x-ray crystal structure
• novel function

Statement dc:rights

• Copyright 2009 Tiit Lukk

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/14751

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/14751