An enzymology and structural biology approach towards elucidation of E. coli fatty acid biosynthesis and its application to the production of bio-renewables

dc.contributor.advisor Adam W. Barb
dc.contributor.author Marcella, Aaron
dc.contributor.department Biochemistry, Biophysics and Molecular Biology
dc.date 2018-08-11T10:28:52.000
dc.date.accessioned 2020-06-30T03:03:21Z
dc.date.available 2020-06-30T03:03:21Z
dc.date.copyright Sun Jan 01 00:00:00 UTC 2017
dc.date.embargo 2001-01-01
dc.date.issued 2017-01-01
dc.description.abstract <p>Prokaryotic fatty acid biosynthesis is an important process to study due to its necessity for cell survival and production of highly reduced carbon chains. A characterization of several proteins involved in E. coli fatty acid biosynthesis is presented. This work provides a method for generating large amounts of pure malonyl acyl-carrier-protein using both the wild type malonyl-CoA:holo-ACP transacylase (FabD) and a variant studied further in our lab. This preparation overcomes the phenomena of a 50:50 mixture of malonyl and acetyl-ACP in the reaction and is crucial for the efficacy of future assays involving malonyl-ACP. A new assay has been developed to measure activity of the FabD enzyme using a fluorescent probe and can be scaled from a rapid to high throughput assay using 96 well plates. FabD variants were studied with the above assay and kinetic constants were determined for several different CoA substrates with enhanced chemical functionality. The structure of holo-ACP synthase in apo and acyl-carrier-protein bound forms was investigated in depth using a combination of X-ray crystallography, NMR spectroscopy, and isothermal titration calorimetry. This work is a comprehensive study of the enzymes involved in the first steps of fatty acid biosynthesis using several biochemical and biophysical techniques.</p>
dc.format.mimetype application/pdf
dc.identifier archive/lib.dr.iastate.edu/etd/15362/
dc.identifier.articleid 6369
dc.identifier.contextkey 11051314
dc.identifier.doi https://doi.org/10.31274/etd-180810-4989
dc.identifier.s3bucket isulib-bepress-aws-west
dc.identifier.submissionpath etd/15362
dc.identifier.uri https://dr.lib.iastate.edu/handle/20.500.12876/29545
dc.language.iso en
dc.source.bitstream archive/lib.dr.iastate.edu/etd/15362/Marcella_iastate_0097E_16340.pdf|||Fri Jan 14 20:39:48 UTC 2022
dc.subject.disciplines Biochemistry
dc.subject.disciplines Biophysics
dc.subject.disciplines Molecular Biology
dc.subject.keywords Fatty acid biosynthesis
dc.subject.keywords Macromolecular crystallography
dc.subject.keywords NMR
dc.title An enzymology and structural biology approach towards elucidation of E. coli fatty acid biosynthesis and its application to the production of bio-renewables
dc.type article
dc.type.genre dissertation
dspace.entity.type Publication
relation.isOrgUnitOfPublication faf0a6cb-16ca-421c-8f48-9fbbd7bc3747
thesis.degree.discipline Biochemistry
thesis.degree.level dissertation
thesis.degree.name Doctor of Philosophy
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