Molecular basis of ene-reductases reactivity and selectivity towards nicotinamide coenzymes

Student thesis: Phd


Understanding the physical basis of enzyme catalysis is critical for deciphering the physiological function of enzymes, and for driving developments in contemporary areas of research, including de novo enzyme design for biotechnology and synthetic biology applications. The increasing knowledge of enzyme structure and mechanisms has led to a shift in the production of fine chemicals from traditional synthetic methods to more environmentally friendly and sustainable approaches. One of the most widely employed chemical reaction in industry for which biocatalytic routes are greatly explored is the asymmetric reduction of activated C=C bonds, which can be catalysed by members of the Old Yellow Enzyme family of ene-reductases. One such member is pentaerythritol tetranitrate reductase (PETNR), a flavin mononucleotide (FMN)-dependent enzyme that uses NADPH (and, less efficiently, NADH) as ancillary hydride donor. Previous kinetic studies of PETNR have inferred that quantum mechanical tunnelling and fast protein dynamics contribute to the enzymatic hydride transfer step from NAD(P)H to the FMN cofactor. Herein, the molecular basis of PETNR reactivity and specificity towards nicotinamide coenzymes is addressed using a wide range of experimental techniques, including mutagenesis, stopped-flow rapid kinetics, X-ray crystallography, temperature dependence/kinetic isotope effect studies of reaction rates, and NMR spectroscopy. 1H, 15N and 13C backbone resonance assignments of PETNR are reported, along with NMR studies evaluating the differences in binding modes of NADPH and NADH coenzymes to PETNR. An investigation of H-transfer mechanism in PETNR through mutagenesis of second sphere 'noncatalytic' residues (L25 and I107) is also presented, and it is the first study probing the role of (rather distal) hydrophobic side chains and dynamics in controlling rates of enzymatic H-transfer catalysed by PETNR. The last section details kinetic studies of rationally designed variants of PETNR and morphinone reductase (MR), another member of the OYE family, which enabled determination of the basis of coenzyme recognition in these two ene-reductases. The approaches developed herein should find wider application in related studies of enzymatic H-transfer reactions and coenzyme specificity studies of other OYE ene-reductases.
Date of Award31 Dec 2019
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorNigel Scrutton (Supervisor), Sam Hay (Supervisor) & Jonathan Waltho (Supervisor)


  • rational enzyme design
  • pentaerythritol tetranitrate reductase
  • flavoenzymes
  • Old Yellow Enzymes
  • backbone resonance assignments
  • morphinone reductase
  • protein crystallography
  • protein dynamics
  • hydride transfer
  • ene-reductases
  • coenzyme specificity
  • protein NMR
  • enzyme kinetics
  • kinetic isotope effects

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