Development of the C-methyltransferases NovO and CouO for biocatalytic Friedel-Crafts alkylation

  • Joanna Christie Sadler

Student thesis: Doctoral Thesis

Abstract

Regiospecific monoalkylation and fluoroalkylation of aromatic compounds is of key importance in medicinal chemistry, yet can be difficult to accomplish chemically. Enzymatic methods may offer a viable alternative to traditional C-C bond forming reactions, such as the Friedel-Crafts alkylation. This thesis describes a number of key advances in the use of two S-adenosyl-L-methionine (SAM) dependent methyltransferases (MTs) NovO (Streptomyces spheroides) and CouO (Streptomyces rishiriensis) as biocatalysts for the Friedel-Crafts alkylation. Firstly, NovO was expressed, purified and crystallised and the X-ray crystal structure of SelMet-NovO was solved to 1.9 Å resolution (PDB accession code: 5MGZ). Additionally, a homology model of CouO (85% sequence identity to NovO) was generated using the NovO structure. Both NovO and CouO are Class I MTs, comprising a Rossmann fold with additional α-helices at the C- and N-termini. Secondly, a catalytic mechanism for NovO was proposed based on the X-ray crystal structure, substrate docking studies, kinetic isotope effect (KIE) data and mutational analysis. This work identified a His-Arg motif to be central to the catalytic mechanism, with initial deprotonation by H120 and stabilisation of the resulting intermediate by R121. Additionally, the KIE data showed that methyl transfer was the rate limiting step. Finally, comparison with CouO identified position 117 in both proteins to be key for mediating the substrate scope of both enzymes. A third major contribution towards the development of NovO and CouO as biocatalysts was preliminary work on their directed evolution (DE) towards a wider substrate scope. Although a more efficient screening or selection strategy will be necessary to increase the substrate scope further, one mutant of NovO (N117M) was identified with higher activity toward 1, 6-dihydroxynaphthalene relative to the WT enzyme, serving as proof of concept for the 'evolvability' of these MTs. The development of a platform for biocatalytic fluoroalkylation using MTs was also explored, with extensive attempts towards the synthesis of a suitable fluoroalkylated cofactor, including the synthesis of novel fluorinated amino acids. A final key contribution towards the development of a biocatalytic Friedel-Crafts alkylation platform was the development of an in situ cofactor synthesis system using the halogenase Sa1L (Salinospora tropica). This was demonstrated on a preparative scale using crude cell lysates (methyl transfer) or purified enzymes (CD3, 13CH3, 13CD3 and Et transfer). This work addressed the issue of cofactor cost and instability associated with SAM dependent MTs.
Date of Award31 Mar 2017
Original languageEnglish
Awarding Institution
  • University Of Strathclyde
SponsorsGlaxo Smithkline (UK) & University of Strathclyde
SupervisorGlenn Burley (Supervisor) & Nick Tomkinson (Supervisor)

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