Investigating intermediates in 6-methylsalicylic acid biosynthesis
type: CreativeWork
description: 6-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 carri…
abstract: 6-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 carri…
author: Potter, Helen Katherine
language: eng
keywords: Biosynthesis, Polyketide
license: https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/321d5bda-336f-4176-9964-63184ba81acf/download
license: http://creativecommons.org/licenses/by-nd/2.0/uk/
date published: 2011-07-12
date modified: 2026-07-22T22:24:32Z
provider: Cambridge University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Cambridge

PropertyValue
property ID: DOI
value: 10.17863/CAM.16283