A structure-based analysis of bioengineering targets in phenyl metabolism for green biotechnology applications

Green biotechnology is the engineering of biological systems for use in environmental and agricultural purposes. Two major areas of environmental biotechnology are bioremediation and biofuel production. Enzyme engineering is often the first approach employed for these objectives because enzymes are the functional units of pathways. Deciphering both the structure and structure-function relationship of enzymes provides a foundation of knowledge to boost rational enzyme engineering efforts.;Bioremediation is the clean-up of industrial wastes with enzymes or organisms. The key to developing these technologies lies in understanding xenobiotic metabolism: the natural breakdown of foreign substances. An important facet of xenobiotic metabolism is glutathione cycling. In prokaryotes and lower eukaryotes, the highly conserved glutathionyl-hydroquinone reductases (GS-HQRs) are essential for this cycling. The structures of GS-HQRs are dimeric and consist of a thioredoxin-like N-terminal domain that binds glutathione and an alpha-helical C-terminal domain that contains a non-specific hydrophobic binding site and a tyrosine network capable of facile proton abstraction and donation. A nucleophilic cysteine stabilized in thiolate form at pH above 7.2, along with the tyrosine network, catalyzes the reductive removal of the glutathione from glutathionyl-hydroquinone conjugates. All essential active site residues are highly conserved in the GS-HQRs from several kingdoms of life.;Bioethanol is the biofuel equivalent of gasoline. A key roadblock to the economical production of bioethanol from agricultural residue is lignin. Lignin is an essential biopolymer for the survival of terrestrial plants but inhibits the release of fermentable sugar polymers from lignocellulosic biomass. Both reduction of lignin content and modification of lignin composition, namely the S:G ratio, in monocots increases sugar yields. A crucial enzyme in the conversion of S to G units is caffeic acid O-methyltransferase (COMT). The structure and kinetic mechanism of Sorghum bicolor's COMT were determined. SbCOMT catalyzes the S-adenosylmethionine-dependent O-methylation of both the 3'- and 5'-hydroxyl of phenylpropanoids. A rapid equilibrium random mechanism for caffeic acid and partial substrate inhibition for 5-hydroxyconiferaldehyde is observed. SbCOMT's structure is highly-conserved among two monocots and one dicot. Additionally, one missense mutation of SbCOMT associated with the favorable brown midrib phenotype was determined to reduce the amount of secondary structure.