Three objectives were studied: determination of the biochemical mechanisms of an Arabidopsis thaliana allylic double bond reductase (AtDBR1) producing stress/defense metabolites, dihydrocinnamyl aldehydes, and the Arabidopsis cinnamyl alcohol dehydrogenase (AtCAD5), as well as exploring the potential of utilizing next-generation sequencing to help identify unknown biochemical steps in formation of the lignan, podophyllotoxin, in Podophyllum species.
Apo and binary structures of AtCAD5 were solved at 2.0 and 2.6 Å resolution, respectively, and ternary complexes were modeled with p-coumaryl aldehyde. A putative proton shuttle mechanism for AtCAD5 involving Thr49, His52 and Asp57 was evaluated, based on a proposed comparable mechanism for horse liver alcohol dehydrogenase. Site-directed mutants of each were prepared with corresponding mutant proteins characterized by kinetic and isothermal titration calorimetry (ITC) analyses. It was established that Thr49 was important in overall catalysis, whereas His52 and Asp57 were not. No evidence was obtained for a putative extended proton relay mechanism in AtCAD5.
Apo, binary and ternary complexes of AtDBR1 were obtained at 2.5 (apo) and 2.8 (binary and ternary) Å resolution, respectively. Analysis of the ternary structure indicated a concerted catalytic mechanism involving hydride transfer to C-7 of p-coumaryl aldehyde (C-3 in 4-HNE), with the Tyr260 hydrogen-bonded to the aldehydic group of p-coumaryl aldehyde and the 2´-OH of nicotine amide ribose. Site-directed mutation of the Tyr260 residue further confirmed an essential role in catalysis through kinetic and ITC analyses.
Illumina-based short reads/next-generation sequencing and bioinformatics analyses, together with a targeted metabolomics approach, of Podophyllum hexandrum and P. peltatum, were used to explore the potential of these technologies to deduce unknown biosynthetic steps to podophyllotoxin. Genes encoding steps in shikimate/chorismate, aromatic amino acid pathways, phenylpropanoid and lignan pathways were assembled, and putative enzymes catalyzing methylene-dioxy bridge formation were identified. Of these, recombinant proteins encoding by two genes CYP719A23 (P. hexandrum ) and CYP719A24 (P. peltatum) were capable of converting (–)-matairesinol into (–)-pluviatolide.
|Advisor:||Tegeder, Mechthild, Lewis, Norman G.|
|Commitee:||Browse, John, Edwards, Gerald, McCubbin, Andrew|
|School:||Washington State University|
|School Location:||United States -- Washington|
|Source:||DAI-B 74/07(E), Dissertation Abstracts International|
|Subjects:||Plant sciences, Biochemistry|
|Keywords:||Dinnamyl alcohol dehydrogenase, Double bond reductase, Lignan, Monolignol|
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