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dc.contributor.advisorLeeper, Finian
dc.contributor.authorPotter, Helen Katherine
dc.date.accessioned2011-10-31T09:51:12Z
dc.date.available2011-10-31T09:51:12Z
dc.date.issued2011-07-12
dc.identifier.urihttp://www.dspace.cam.ac.uk/handle/1810/239407
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/239407
dc.description.abstract6-Methylsalicylic acid (6-MSA) is one of the oldest known polyketides. It is synthesised in vivo by the polyketide synthase 6-methylsalicylic acid synthase (6-MSAS), a multifunctional enzyme which uses its active sites iteratively. The stereochemistry of the hydroxyl produced from the single ketoreduction, as well as the order of dehydration, cyclisation and aromatisation steps, remain cryptic, despite extensive study. Holo 6-MSAS was heterologously expressed in E. coli and purified in two steps. A non-hydrolysable carba(dethia)malonyl-N-acetylcysteamine analogue was synthesised and used to off-load enzyme-bound intermediates from 6-MSAS. In assays with acetyl-CoA and acetoacetyl-CoA alone, diketide and triketide intermediates were off-loaded and detected by HPLC-HR-ESI-MS. In the presence of NADPH, the off-loaded triketide was reduced by the ketoreductase domain of 6-MSAS. A potential dehydrated intermediate was also observed. The dehydratase domain of 6-MSAS has recently been reassigned as a thioester hydrolase. To test this theory, the catalytic histidine residue in 6-MSAS was mutated to an alanine and the abolition of production of 6-MSA in vivo was observed. Mutated 6-MSAS was still able to produce the shunt product triacetic acid lactone. Incubation of mutated 6-MSAS with acetyl-CoA, malonyl-CoA, NADPH and carba(dethia)malonyl-N-acetylcysteamine saw only the off-loading of diketide and triketide analogues. To investigate the stereochemistry of ketoreduction in 6-MSA biosynthesis, steps were made to synthesise the resolved diastereomeric reduced-triketide CoAs which would be the substrates for the ketoreductase domain. Attempts to phosphopantetheinylate apo 6-MSAS in vitro with three different phosphopantetheinyltransferases were unsuccessful. Limited proteolysis of both holo and apo 6-MSAS found that the apo synthase rapidly lost a C-terminal fragment while holo 6-MSAS was much more stable under the same conditions. Attempts were made to express the acyl carrier protein domain from 6-MSAS to overcome these problems. These experiments represent the first use of the non-hydrolysable analogue methodology in a Type I iterative polyketide synthase and provide a framework for future experiments investigating intermediates in the biosynthesis of 6-MSA.en_GB
dc.language.isoenen_GB
dc.rightsAttribution-NoDerivs 2.0 UK: England & Walesen
dc.rights.urihttp://creativecommons.org/licenses/by-nd/2.0/uk/en
dc.subjectBiosynthesisen_GB
dc.subjectPolyketideen_GB
dc.titleInvestigating intermediates in 6-methylsalicylic acid biosynthesisen_GB
dc.typeThesisen_GB
dc.type.qualificationleveldoctoralen_GB
dc.type.qualificationnamePhDen_GB
dc.publisher.institutionUniversity of Cambridgeen_GB
dc.publisher.departmentDepartment of Chemistryen_GB
dc.identifier.doi10.17863/CAM.16283


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Attribution-NoDerivs 2.0 UK: England & Wales
Except where otherwise noted, this item's licence is described as Attribution-NoDerivs 2.0 UK: England & Wales