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Title:Functional assignments in the enolase superfamily: investigations of two divergent groups of D-galacturonate dehydratases and galactarate dehydratase-III
Author(s):Groninger-Poe, Fiona
Director of Research:Gerlt, John A.
Doctoral Committee Chair(s):Gerlt, John A.
Doctoral Committee Member(s):Cronan, John E.; Fratti, Rutilio A.; Huang, Raven H.
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):enolase superfamily
D-galacturonate dehydratase
m-galactarate dehydratase
Abstract:More than a decade after the genomic age, full genome sequencing is cost-effective and fast, allowing for the deposit of an ever increasing number of DNA sequences. New fields have arisen from this availability of genomic information, and the way we think about biochemistry and enzymology has been transformed. Unfortunately, there is no robust method for accurately determining the functions of enzymes encoded by these sequences that matches the speed in which genomes are deposited into public databases. Functional assignment of enzymes remains of utmost importance in understanding microbial metabolism and has applications in agriculture by examining bacterial plant pathogen metabolism and additionally in human health by providing metabolic context to the human gut microbiome. To aid in the functional identification of proteins, enzymes can be grouped into superfamilies which share common structural motifs as well as mechanistic features. To this end, the enolase superfamily is an excellent model system for functional assignment because more than half of the members still lack functional identification. Structurally, these enzymes contain substrate specificity residues in the N-terminal capping domain and catalytic residues in the C-terminal barrel domain. Mechanistically, each enzyme catalyzes the abstraction of a proton alpha to a 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, laying the groundwork for functional characterization of unknown members. In this thesis, I address the problem of functional assignment by utilizing computational, structural, biochemical, and microbiological techniques to assign previously undiscovered functions to three groups of enzymes in the enolase superfamily that were previously unknown: the Microscilla group of D-galacturonate dehydratases, the Geobacillus group of D-galacturonate dehydratases, and galactarate dehydratase-III from Agrobacterium tumefaciens strain C58. Although the two groups of D-galacturonate dehydratases produce an identical product, they act through different mechanisms and have different structural elements. The functional assignments of these enzymes contribute to our understanding of the potential mechanisms and functions possible in this superfamily. The Microscilla group of D-galacturonate dehydratases (GalurDs) from Microscilla species PRE-1, Streptomyces coelicolor A3(2), Saccarophagus degradans 2-40, Pseudoalteromonas atlantica T6c and others utilize D-galacturonate to produce 5-keto-4-deoxygalacturonate in dehydration reaction consistent with known acid-sugar dehydratases in the enolase superfamily. These enzymes house a KxK motif on the second beta strand and a H/D dyad at the seventh and sixth beta strand in the barrel domain. These enzymes are found in organisms that degrade agar. In Microscilla species PRE-1 the GalurD gene is encoded on a plasmid along with other genes involved in agar degradation implying that GalurD could be contributing to the metabolism of agar. The Geobacillus group of D-galacturonate dehydratases from Geobacillus and Paenibacillus are structurally divergent from the abovementioned Microscilla group of GalurDs; unlike other acid-sugar dehydratases in the enolase superfamily, the Geobacillus GalurD contains an unusual second magnesium ion near the fourth beta strand of the barrel. Upon investigation, we believe this second magnesium is not catalytic and is rather an artifact of crystallization. Although these enzymes perform the same reaction as GalurDs from Microscilla, the Geobacillus group has a different mechanism and different sequence and is thus a separate group of GalurDs in the enolase superfamily. Galactarate dehydratase-III (GalrD-III) from Agrobacterium tumefaciens strain C58 is unlike previously reported galactarate dehydratases in the enolase superfamily in terms of sequence and substrate specificity: GalrD-III dehydrates galactarate with catalytic residues similar to galactonate dehydratases (galactonate dehydratases do not dehydrate galactarate, nor do galactarate dehydratases dehydrate galactonate) making this reaction unique in the enolase superfamily. Furthermore, GalrD-III dehydratases D-galacturonate by abstracting the proton located alpha to the aldehyde, producing a diketo product which undergoes a Benzil rearrangement to yield either 3-deoxy-D-xylo-hexarate or 3-deoxy-D-lyxo-hexarate (stereochemistry at C2 is uncertain), a compound not found in any known metabolic pathway. This is a novel mechanistic step and product in the enolase superfamily which prompts us to reexamine the possible substrates that can be utilized by enolase superfamily members. In providing these functional assignments to these groups of enzymes, we are not only able to extend these functions to other enolase superfamily members sharing the appropriate structural characteristics, but we can also expand our screening libraries and methods to incorporate aldose sugars; these have been previously overlooked as a possible substrate for sugar dehydratases in this superfamily. Thus, this work contributes to the problem of functional assignment by illuminating additional mechanistic and functional possibilities in the enolase superfamily.
Issue Date:2014-09-16
Rights Information:Copyright 2014 Fiona Groninger-Poe
Date Available in IDEALS:2014-09-16
Date Deposited:2014-08

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