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Title:Functional and physiological discovery in the mannonate dehydratase subgroup of the enolase superfamily
Author(s):Wichelecki, Daniel
Director of Research:van der Donk, Wilfred A.; Cronan, John E.; Silverman, Scott K.
Doctoral Committee Chair(s):Gerlt, John A.
Doctoral Committee Member(s):van der Donk, Wilfred A.; Cronan, John E.; Silverman, Scott K.
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Enzyme Function Initiative
mannonate dehydratase
gluconate dehydratase
Reverse Thymidylate Synthase (rTS)
Enolase Superfamily Member 1 (ENOSF1)
functional discovery
physiological discovery
enzyme evolution
enolase superfamily
Abstract:In the current post-genomic world, the exponential amassing of protein sequences is overwhelming the scientific community’s ability to experimentally assign each protein’s function. The use of automated, homology-based annotations has allowed a reprieve from this efflux of data, but has led to widespread misannotation and nonannotation in protein sequence databases. This dissertation details the functional and physiological characterization of the mannonate dehydratase subgroup (ManD) of the enolase superfamily (ENS). The outcome affirms the dangers of homology-based annotations while discovering novel metabolic pathways. Furthermore, the experimental verification of these pathways (in vitro and in vivo) has provided a platform to test the general strategies for improved functional and metabolic characterization being developed by the Enzyme Function Initiative (EFI). Prior to this study, one member of the ManD subgroup had been characterized and was shown to dehydrate D-mannonate to 2-keto-3-deoxy-D-gluconate. Forty-two additional members of the ManD, selected from across the sequence space of the subgroup, were screened for activity and kinetic constants were determined. The members of the once isofunctional subgroup were found to differ in both catalytic efficiency and substrate specificity: 1) high efficiency (kcat/KM = 103 to 104 M-1s-1) dehydration of D mannonate, 2) low efficiency (kcat/KM = 101 to 102 M-1s-1) dehydration of D-mannonate and/or D-gluconate, and 3) no-activity with either D-mannonate or D gluconate (or any other acid sugar tested). The novel D-gluconate activity in this subgroup was investigated, and the mechanism of its enzymatic action was discovered. Physiologically, D mannonate dehydration is essential to D-glucuronate metabolism. The D mannonate dehydratase, UxuA, is not a member of the ENS. No uxuA genes are found in the genome of organisms with high efficiency ManDs. Through in vitro characterization and in vivo verification, a high efficiency ManD was discovered in Caulobacter crescentus CB15 that fulfills the same physiological role as UxuA and is an example of convergent evolution. The genomes of organisms with low efficiency members of the ManD subgroup generally have the uxuA gene. Therefore, they likely fulfill a different physiological role than the high efficiency ManDs. Their in vitro characterization and in vivo functional verification lead to the discovery of a novel L-gulonate metabolic pathway in Chromohalobacter salexigens DMS3043 where L-gulonate is converted to D-mannonate by a dehydrogenase and a reductase. While the low efficiency ManD found in C. salexigens is not metabolically essential to this pathway, its presence lead to the discovery of the pathway. Similar methods in Salmonella enterica subsp. enterica serovar Enteritidis str. P125109 lead to the discovery a novel L-idonate pathway where L-idonate is converted to D-gluconate by two dehydrogenases and then dehydrated (the traditional pathway phosphorylates D-gluconate). This pathway directly involves a low efficiency GlcD that is a member of the ManD subgroup and raises interesting questions about the physiological role of low efficiency enzymes and redundant pathways. As a whole, this dissertation displays how function diverges as sequence diverges while laying bare the dangers of annotation via homology; concurrently, it demonstrates how the continually advancing assignment strategies of the EFI can be used to discover new enzymatic functions and metabolic pathways.
Issue Date:2015-01-21
Rights Information:Copyright 2014 Daniel Wichelecki. Biochemistry owns the publications which were adapted to be chapters 2, 3, 4, and 5. Permission was acquired to reprint these.
Date Available in IDEALS:2015-01-21
Date Deposited:2014-12

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