Engineering Alkyltransferases as Synthetic Tools for Biocatalysis

Abstract

Methylation and alkylation are often key steps in the synthesis or modification of active pharmaceutical ingredients (APIs). Using a biocatalytic approach to perform this step offers a number of advantages over traditional chemical methods; in addition to avoiding toxic reagents and the environmentally harmful effects associated with chemical alkylation, biocatalysis can be highly chemo-, regio- and stereo-selective. This project has therefore focused on using S-adenosyl-methionine (SAM)-dependent methyltransferases (MTases) to transfer either methyl or alkyl groups enzymatically. Specifically, this work has focused on utilising the recently discovered biological cofactor carboxy-S-adenosyl methionine (CxSAM) to transfer its carboxy-methyl group to a diverse range of substrates. SAM-dependent MTases are a class of biologically important enzymes responsible for catalysing the transfer of a methyl group from the cofactor SAM to proteins, nucleic acids and various small biomolecules. A number of SAM-dependent MTases, however, exhibit sufficient promiscuity to accept SAM analogues with alternative functional groups in place of the S-methyl group or can be engineered to do so. This hence provides a useful biocatalytic tool to transfer a range of chemical moieties. In this study we utilise the E. coli enzyme CmoA, that was recently discovered to catalyse the conversion of SAM to a novel SAM analogue, in tandem with a MTase which is promiscuous in accepting the alternative cofactor. Three class I MTases were shown to accept CxSAM: catechol-O-methyltransferase (COMT) from Rattus norvegicus, coclaurine N-methyltransferase (CNMT) from Coptis japonica, and tetrahydroprotoberberine cis-N-methyltransferase (TNMT) from Papaver somniferum can all utilise CxSAM to catalyse a carboxy-methyl transfer reaction, with catechol (COMT) and tetrahydroisoquinoline (CNMT, TNMT) type substrates. Of particular interest are the tetrahydroisoquinoline based compounds, which possess a diverse range of therapeutic and pharmacological activities. A site-directed mutagenesis approach was therefore applied to the CNMT enzyme, with the aim to improve activity with the non-native cofactor, CxSAM, and also improve the cofactor selectivity of CNMT towards CxSAM over SAM.

Date of Award	1 Aug 2018
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Jason Micklefield (Supervisor) & Nicholas Turner (Supervisor)