Dissertation
STRUCTURAL, BIOCHEMICAL CHARACTERIZATION OF ENZYMES RELATED TO RENEWABLE ENERGY AND ENVIRONMENTAL REMEDIATION
Doctor of Philosophy (PhD), Washington State University
01/2017
Handle:
https://hdl.handle.net/2376/117255
Abstract
A structural analysis of an enzyme reveals enzymatic mechanism and allows guided modification on an enzyme or design a substrate for a desired outcome, due to the structure-activity relationship. To develop sustainable biochemical solutions for environmental management, a thorough characterization of participating enzymes must be preceded. In this study, enzymes potentially lower the cost of bioethanol production, and bioremediation of ethylenediaminetetraacetate (EDTA) are characterized.
Phenylalanine ammonia-lyase (PAL) and cinnamyl alcohol dehydrogenase (CAD) catalyze the first and the last steps in monolignol biosynthesis, respectively. PAL initiate the pathway by catalyzing elimination of ammonia from L-phenylalanine/tyrosine to give trans-cinnamate/p-coumarate, whereas CAD catalyzes the reduction of trans-hydroxycinnamaldehyde family to yield monolignols. Here, we present the first crystal structure of a monocotyledonous PAL and CAD from Sorghum bicolor, a strategic plant for bioenergy production, with identified critical residues and explained substrate specificity. SbPAL1 displayed a unique active-site conformation where His123 sidechain dictated binding mode of substrates, conferring the deamination reaction through either Friedel–Crafts or Hofmann elimination. H123F-SbPAL1 mutant showed 6.2x PAL activity enhancement that could establish the engineering basis for potential benefits on both silage/forage quality as well as production of renewable fuels and chemicals from plant biomass. Also, a structure guided CAD double mutant, L119W/G301F-SbCAD4, displayed substrate-preference favorable to bioethanol production, with higher catalytic efficiency than wild-type SbCADs. Thus, L119W/G301F-SbCAD4 in plants could produce a phenotype that is more amenable to biomass processing.
EDTA monooxygenase (EmoA) forms a two-component flavin-dependent monooxygenases (TCFDM) with its oxidoreductase partner (EmoB) to degrade EDTA, a ubiquitous organic pollutant. Although EmoA has been identified from Chelativorans sp. BNC1, its catalytic mechanism is unknown. Crystal structures of EmoA revealed a domain-like insertion into a TIM-barrel, which may serve as a flexible lid for the active site. Docking of MgEDTA2+ into EmoA identified an intricate hydrogen bond network connected to Tyr71, lowering its pKa. Tyr71, along with nearby Glu70 and a peroxy flavin, facilitates a keto-enol transition of the leaving acetyl group of EDTA. Further, for the first time, the physical interaction between TCFDM was observed.
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Details
- Title
- STRUCTURAL, BIOCHEMICAL CHARACTERIZATION OF ENZYMES RELATED TO RENEWABLE ENERGY AND ENVIRONMENTAL REMEDIATION
- Creators
- SE-YOUNG JUN
- Contributors
- ChulHee Kang (Advisor)Clifford E Berkman (Committee Member)James A Brozik (Committee Member)Jeffrey P Jones (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- Department of Chemistry
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Number of pages
- 155
- Identifiers
- 99900581625601842
- Language
- English
- Resource Type
- Dissertation