Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.588423
Title: Engineering carboxymethylproline synthases towards the biosynthetic productions of carbapenem antibiotics
Author: Gómez Castellanos, José Rubén
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2013
Availability of Full Text:
Access through EThOS:
Full text unavailable from EThOS. Restricted access.
Access through Institution:
Abstract:
Mechanistic and biocatalytic studies of two carboxymethylproline synthases (CMPSs), CarB and ThnE, members of the crotonase superfamily of enzymes, both in isolation and in conjunction with the activity of the crotonyl-CoA carboxylase/reductase (Ccr) the malonyl-CoA synthetase (MatB) and the methylmalonyl-CoA epimerase (MCE) are presented. Protein engineering studies on carboxymethylproline synthases aimed at enabling stereoselective C–C bond formation leading to N-heterocycles via control of trisubstituted enolate intermediates were carried out. Active site substitutions, including at the oxyanion binding site, enabled the production of substituted N-heterocycles in high diastereomeric excesses via stereocontrolled enolate formation and reaction. The biocatalytic promiscuity of malonyl-CoA ligase and the stereoselectivity of crotonyl–CoA carboxylase/reductase were successfully coupled to the selective tri- substituted enolate forming capacity of engineered carboxymethylproline synthases for the preparation of functionalized 5- and 6-membered N-heterocycles substituted with a variety of alkyl side chains at the C-5/C-6 positions at high diastereomeric excess. The effect of methylmalonyl-CoA epimerase on the diastereoselectivity of the carboxymethylproline synthase-catalysed enolated alkylation was also demonstrated. The results illustrate the utility of the crotonase superfamily of enzymes for stereoselective biocatalysis and demonstrate the power of coupled enzyme systems to enhance diastereoselectivity and to expand the range of accepted substrates.
Supervisor: Schofield, Christopher J. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.588423  DOI: Not available
Keywords: Chemical biology ; carboxymethylproline synthases ; combinatorial biosynthesis ; carbapenems
Share: