Dissertation
BIOSYNTHESIS AND MODIFICATION OF UNUSUAL AMINO ACIDS THROUGH ENZYME-CATALYZED CHEMISTRY
Doctor of Philosophy (PhD), Washington State University
01/2014
Handle:
https://hdl.handle.net/2376/5431
Abstract
Members of the radical S-adenosyl-L-methionine (SAM) family of enzymes catalyze reactions that would normally be biologically impossible by utilizing SAM as a source of a primary radical. Many of the reactions catalyzed by these enzymes change the traditional paradigms for biological chemistry. The putative radical SAM enzymes PhpK and PylB both catalyze unusual amino acid modifications that have remained largely uncharacterized over the past 20 years. PhpK catalyzes methyl transfer to an electrophilic phosphinate, completing the only known naturally-occurring carbon-phosphorus-carbon linkage. We developed methodology to isolate, purify, and begin in vitro characterization of PhpK. We found PhpK was methylcobalamin-dependent, transferring the methyl group from methylcobalamin directly to the final product. The enzyme used a CxxxCxxC bound four-iron, four-sulfur cluster together with SAM to catalyze P-methylation, consistent with known radical SAM mechanisms. PylB catalyzes the first step in the biosynthesis of the 22nd genetically-encoded amino acid pyrrolysine. It performs a lysine isomerization to form 3-methyl-D-ornithine. Since the protons at C-4 have a pKa~50, the reaction is undoubtedly catalyzed by a radical mechanism. We have begun developing methods for purification and assays of PylB to study the mechanistic details of this reaction. These studies set the stage for characterization of these radical SAM enzymes and future engineering efforts towards industrial/commercial applications for the reactions they catalyze.
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Details
- Title
- BIOSYNTHESIS AND MODIFICATION OF UNUSUAL AMINO ACIDS THROUGH ENZYME-CATALYZED CHEMISTRY
- Creators
- Williard J Werner
- Contributors
- Susan C Wang (Advisor)Lisa M Gloss (Committee Member)Philip F Mixter (Committee Member)Michael J Smerdon (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- Molecular Biosciences, School of
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Number of pages
- 194
- Identifiers
- 99900581733301842
- Language
- English
- Resource Type
- Dissertation