Directed evolution and structure-function studies of oxidosqualene cyclases

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dc.contributor.advisorMatsuda, Seiichi P. T.en_US
dc.creatorSegura, Michael Joseph Roberten_US
dc.date.accessioned2009-06-04T08:11:42Zen_US
dc.date.available2009-06-04T08:11:42Zen_US
dc.date.issued2003en_US
dc.description.abstractAlthough oxidosqualene cyclase reaction mechanisms have been established, how these enzymes promote specific transformations, generate product diversity, and achieve product specificity is only partially understood. Protein mutagenesis experiments described herein have identified catalytically important residues involved in deprotonation and cyclization steps of oxidosqualene cyclase catalysis. Residues influencing specific deprotonation in lanosterol and cycloartenol synthase were identified. The Saccharomyces lanosterol synthase Thr384Tyr mutation compromised product specificity and caused the formation of the novel lanosta-24-ene-3beta,9alpha-diol, a compound previously unidentified in nature. Furthermore, the Arabidopsis thaliana cycloartenol synthase His477 position was identified by random mutagenesis. Interestingly, subtle amino acid changes at this position show dramatic and different influences on product structure; the His477Asn mutant makes predominantly lanosterol, whereas the His477Gln mutant makes mostly parkeol. The AthCAS1 His477Asn and His477Gln mutants are currently the most accurate lanosterol and parkeol synthases, respectively, generated by protein mutagenesis. AthCAS1 His477 mutations were combined with other catalytically important mutations (AthCAS1 Tyr410Thr Ile481Val) to see how the combined mutations would interact to influence product structure. Surprisingly, the His477 mutations did not influence catalysis when combined with the other mutations. The catalytic behavior of these AthCAS1 triple mutants is the first demonstration of dominant and recessive properties of catalytically important oxidosqualene cyclase mutations. DNA shuffling of cycloartenol and lupeol synthase was undertaken to determine what residues or motifs control substrate folding, a reaction step that has important consequences for product structure. Hybrid enzymes isolated after one round of DNA shuffling revealed that the N- and C-terminal ends (115 and 140 a.a., respectively) of cycloartenol synthase do not contain residues that are required for protosteryl cation formation or cyclopropyl ring formation. Those hybrids possessing lupeol synthase sequence at the termini but retaining internal cycloartenol synthase sequence maintained cycloartenol biosynthetic ability, demonstrating that catalytic components required for cycloartenol biosynthesis are located in the internal portions of the enzyme sequence. Site-directed mutagenesis and chimeragenesis experiments further defined catalytically important regions indicated by DNA shuffling experiments. In addition, this thesis describes the complete characterization of the Arabidopsis lupeol synthase and the construction of a novel yeast strain that possesses a plant sterol biosynthetic pathway.en_US
dc.format.extent109 p.en_US
dc.format.mimetypeapplication/pdfen_US
dc.identifier.callnoTHESIS BIOCHEM. 2003 SEGURAen_US
dc.identifier.citationSegura, Michael Joseph Robert. "Directed evolution and structure-function studies of oxidosqualene cyclases." (2003) Diss., Rice University. <a href="https://hdl.handle.net/1911/18561">https://hdl.handle.net/1911/18561</a>.en_US
dc.identifier.urihttps://hdl.handle.net/1911/18561en_US
dc.language.isoengen_US
dc.rightsCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.en_US
dc.subjectMolecular biologyen_US
dc.subjectBiochemistryen_US
dc.subjectOrganic chemistryen_US
dc.titleDirected evolution and structure-function studies of oxidosqualene cyclasesen_US
dc.typeThesisen_US
dc.type.materialTexten_US
thesis.degree.departmentBiochemistry and Cell Biologyen_US
thesis.degree.disciplineNatural Sciencesen_US
thesis.degree.grantorRice Universityen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophyen_US
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