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dc.rights.licenseRestricted to current Rensselaer faculty, staff and students. Access inquiries may be directed to the Rensselaer Libraries.
dc.contributorDordick, Jonathan S.
dc.contributorBystroff, Christopher, 1960-
dc.contributorKoffas, Mattheos A. G.
dc.contributorMaxwell, Patrick H.
dc.contributor.authorLidsky, Israel
dc.date.accessioned2021-11-03T08:28:16Z
dc.date.available2021-11-03T08:28:16Z
dc.date.created2015-10-01T11:33:09Z
dc.date.issued2015-08
dc.identifier.urihttps://hdl.handle.net/20.500.13015/1531
dc.descriptionAugust 2015
dc.descriptionSchool of Science
dc.description.abstractCis-prenyltransferases (CPT) are a class of enzymes that biocatalyze terpenoid-pathway cis-1,4-polyisoprenoids (CP) from 5-carbon isoprenoid precursors. CPT products range from bacterial peptidoglycan cell wall lipid shuttles, to dolichol precursors for mammalian protein glycosylation. Some plants use a putative rubber transferase CPT to produce natural rubber in plant latex. CPTs are difficult to study, as the isoprenoid products tend to be large and hydrophobic. Additionally, CPs are not easily UV-Vis active. As part of this work, we use a CPT that produces CPs of up to 11 isoprene units in length as a model system. Our model CPT is Micrococcus luteus undecaprenyl pyrophosphate synthase (UppS), which forms 55-carbon undecaprenyl pyrophosphate (UPP) product from 5-carbon isopentenyl pyrophosphate (IPP) and 15-carbon farnesyl pyrophosphate (FPP) substrates. Typically, CPTs or their products are membrane-associated, as opposed to their hydrophilic IPP substrate. Literature suggests that, for UppS aqueous reactions, product release is rate limiting in the absence of the surfactant Triton X-100.
dc.description.abstractTypically, CPTs or their products are membrane-associated, as opposed to their hydrophilic IPP and FPP substrates. Surfactant may be interacting with UppS in a manner similar to in vivo lipid bodies, as the AOTPS has hydrophilic and hydrophobic solvents interacting at an interface. We hypothesize that an aqueous-organic two-phase system (AOTPS) with equal parts aqueous buffer and organic solvent will mimic in vivo lipid bodies and allow for CPT activity. We employed liquid-chromatography mass-spectrometry to measure substrate consumption and UPP formation from non-derivatized samples. The research presented herein shows that a UppS AOTPS reaction cannot fully replace the requirement for surfactants, but demonstrates that an AOTPS can enhance product-formation for reactions containing surfactant. We also show that different organic solvents in an AOTPS will have an effect on UppS product formation. Liquid-chromatography mass-spectrometry is used as the method for measuring substrate consumption and UPP formation from non-derivatized samples. These data suggests that UppS is a good model for studying CPT activity, which may be used as a surrogate model for studying rubber transferases.
dc.language.isoENG
dc.publisherRensselaer Polytechnic Institute, Troy, NY
dc.relation.ispartofRensselaer Theses and Dissertations Online Collection
dc.subjectBiology
dc.titleAqueous-organic two-phase systems enhance activity of undecaprenyl pyrophosphate synthase
dc.typeElectronic thesis
dc.typeThesis
dc.digitool.pid176729
dc.digitool.pid176730
dc.digitool.pid176731
dc.rights.holderThis electronic version is a licensed copy owned by Rensselaer Polytechnic Institute, Troy, NY. Copyright of original work retained by author.
dc.description.degreePhD
dc.relation.departmentDept. of Biological Sciences


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