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Title:Mining the enolase superfamily for new functions: investigations of D-glucarate dehydratase related proteins (GlucDRP) and L-lyxonate dehydratase proteins (LyxD)
Author(s):Ghasem pur, Salehe
Director of Research:Gerlt, John A.
Doctoral Committee Chair(s):Gerlt, John A.
Doctoral Committee Member(s):van der Donk, Wilfred A.; Morrissey, James H.; Tajkhorshid, Emad
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Enolase
L-lyxonate
Heterodimer
Heterospecies
D-Glucarate
Abstract:Genomic era begins with development of sequencing methods. Genome sequencing is now cost-effective and fast, giving rise to increasing amounts of genomic data. However, the function of 1% of the deposited sequences have been experimentally characterized. There is no robust method of functional assignment for these sequences. Functional assignments are now performed using a variety of software tools to utilize the known biochemical data of characterized proteins to annotate similar sequences in genome databases. With this large scale automatic annotations of genome databases, annotations were transferred from homologs regardless of their reliability which result in propagation of errors and transfer misleading information to scientific community. Nevertheless, annotating the homologs within a superfamily is a valid approach. To this end, the enolase superfamily is an excellent model system for functional assignment. Structurally, this superfamily contain substrate specificity residues in the N-terminal capping domain and catalytic residues in the C-terminal barrel domain. These proteins with the common structural fold have the ability to abstract a proton α to carboxylate on the substrate before proceeding to dehydration, epimerization, deamination, racemization or cycloisomerization. There have been enough studies of this superfamily to provide valuable insight into the types of reactions performed based on catalytic residues and substrate specificity residues, establishing basis for functional characterization of unknown members. In this thesis, I discuss my efforts on characterizing an enolase superfamily member: D- glucarate dehydratase related protein (GlucDRP). GlucDRP share more than 60% sequence identity with GlucD, a well-characterized protein. I showed that there is a protein-protein interaction between GlucD and GlucDRP which form a detectable heterospecies. The kinetics of heterospecies, isolated from wild type E. coli, was equal to an average of the activity of a GlucD and a GlucDRP. Additionally, to determine what percent of GlucDRPs involves in the formation of the heterospecies, a hexahistidine-tag was introduces upstream of the GlucDRP gene in the E. coli chromosome and it was shown that more than half of expressed GlucDRP forms heterospecies. Finally, the three-dimensional crystal structure of the heterospecies was determined which confirmed the formation of α2/β2 tetramers of GlucD and GlucDRP. At a BLAST e-value of 10-175, three clusters of GlucDRPs segregated from the authentic GlucD. Structural alignments of GlucD and GlucDRP proteins showed good superposition between these structures except in 100s loop, which interacts with the substrate and closes the active site upon entrance of the substrates. This structural differences may be responsible for differences in catalytic activity. I investigated the protein-protein interaction between GlucD and GlucDRP from the three GlucDRP clusters in three organisms: Burkholderia cepacia, Actinobacillus succinogenes 130Z, and Ralstonia pickettii 12j and the formation of heterospecies in these proteins were confirmed. Additionally, I showed that GlucDRP promotes faster growth on glucarate media. D-glucarate is a component of urine, and as an occasional colonizer of the bladder and urinary tract, E. coli might have evolved mechanisms to rapidly catabolize a less favored source of energy like D-glucarate and outcompete other resident microorganisms. In this thesis, I assigned a novel function, L-lyxonate dehydratase activity, to a mandelate racemase (MR) subgroup of the enolase superfamily. In vitro and in vivo data were combined here to show that the dehydration of L-lyxonate is the biological role of the enzymes in this protein family. Through in vitro experiment, catalytic efficiency of ~ 104 M-1s-1 was measured for L-lyxonate dehydratase protein. In vivo growth studies revealed that L-lyxonate is a carbon source for Pseudomonas aeruginosa and transcriptome analysis showed that the L-lyxonate dehydratase gene along with some neighboring genes were expressed in L-lyxonate grown cells. The neighboring genes were cloned and purified to be tested on the L-lyxonate degradation intermediates and it was shown that upon dehydration of L-lyxonate and formation of 2-keto-3-deoxy-L-lyxonate, a second dehydratase act to convert the product to yield α-ketoglutarate semialdehyde. In the final step a dehydrogenase oxidizes α-ketoglutarate semialdehyde to α-ketoglutarate, an intermediate in the citric acid cycle. Mutational studies revealed that L-lyxonate dehydratase proteins possess a catalytic His-Asp dyad at the end of the seventh and six β-strands of the (β/α)7β-barrel domain and a KxR motif at the end of second β-strand. This is the first example of an L-lyxonate dehydratase in the enolase superfamily and the first example of a pathway for degradation of L-lyxonate.
Issue Date:2015-01-21
URI:http://hdl.handle.net/2142/73056
Rights Information:Copyright 2014 Salehe Ghasem pur
Date Available in IDEALS:2015-01-21
Date Deposited:2014-12


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