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University of Illinois – Urbana-Champaign

1. Ghasem pur, Salehe. Mining the enolase superfamily for new functions: investigations of D-glucarate dehydratase related proteins (GlucDRP) and L-lyxonate dehydratase proteins (LyxD).

Degree: PhD, 0318, 2015, University of Illinois – Urbana-Champaign

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… Advisors/Committee Members: Gerlt, John A. (advisor), Gerlt, John A. (Committee Chair), van der Donk, Wilfred A. (committee member), Morrissey, James H. (committee member), Tajkhorshid, Emad (committee member).

Subjects/Keywords: Enolase; L-lyxonate; Heterodimer; Heterospecies; D-Glucarate

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Ghasem pur, S. (2015). Mining the enolase superfamily for new functions: investigations of D-glucarate dehydratase related proteins (GlucDRP) and L-lyxonate dehydratase proteins (LyxD). (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/73056

Chicago Manual of Style (16th Edition):

Ghasem pur, Salehe. “Mining the enolase superfamily for new functions: investigations of D-glucarate dehydratase related proteins (GlucDRP) and L-lyxonate dehydratase proteins (LyxD).” 2015. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed July 17, 2019. http://hdl.handle.net/2142/73056.

MLA Handbook (7th Edition):

Ghasem pur, Salehe. “Mining the enolase superfamily for new functions: investigations of D-glucarate dehydratase related proteins (GlucDRP) and L-lyxonate dehydratase proteins (LyxD).” 2015. Web. 17 Jul 2019.

Vancouver:

Ghasem pur S. Mining the enolase superfamily for new functions: investigations of D-glucarate dehydratase related proteins (GlucDRP) and L-lyxonate dehydratase proteins (LyxD). [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2015. [cited 2019 Jul 17]. Available from: http://hdl.handle.net/2142/73056.

Council of Science Editors:

Ghasem pur S. Mining the enolase superfamily for new functions: investigations of D-glucarate dehydratase related proteins (GlucDRP) and L-lyxonate dehydratase proteins (LyxD). [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2015. Available from: http://hdl.handle.net/2142/73056

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