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Title: A promiscuous and catalytically proficient de novo carbene transferase
Author: Stenner, Richard A.
ISNI:       0000 0004 9347 1935
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2020
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Biocatalysis is the use of biological materials, such as whole cells or enzymes, to perform chemical transformations. One of the major obstacles limiting the widespread adoption of biocatalytic methodologies in organic synthesis is the restricted number of chemical transformations enzymes are capable of performing. To address this, the field of rational design utilises the techniques of molecule biology, such as mutagenesis, molecular cloning, and recombinant protein technology, to engineer novel enzymes with abiological activity. Rational design can be split into two complementary fields: protein redesign and de novo design. Protein redesign involves identifying a suitable, naturally occurring protein scaffold to function as a molecular blueprint into which desired functionality can be engineered. De novo design is a bottom-up approach, in which novel proteins are designed from scratch using first principles. The recent success in engineering carbene transferases using artificial cytochrome P450 and globin scaffolds has expanded the repertoire of biocatalysis into carbene transfer chemistry. The reactive intermediate in all the reported artificial carbene transferases is a heme-localised electrophilic metallocarbenoid intermediate. C45 is a tetra-α-helical c-type heme-containing de novo protein which functions as a proficient oxidoreductase. The first question raised is whether C45 is capable of forming a metallocarbenoid intermediate, and, if it can, can C45 catalyst carbene transfer reactions with an assortment of suitable substrates, such as olefins (cyclopropanation), X-H α-bonds (X-H insertions), aldehydes (carbonyl olefinations), and nitrogen-containing heteroaromatics (homologous ring expansions)? C45 was screened for metallocarbenoid formation activity using spectroscopic techniques and mass spectrometry, and its reactivity was quantified using a variety of small molecule analytical techniques. In addition, the inherent simplicity and evolvability of C45 rendered the protein scaffold a perfect blueprint for direct evolution, and novel stereocomplementary tetra-α-helical c-type heme-containing maquettes were developed, biophysically characterised, and enzymatically evaluated. Another significant obstacle hindering the widespread adopting of biocatalytic methodologies in organic synthesis is the limited number of operating conditions hospitable to enzymes. In particular, the presence of organic solvents is often completely detrimental to enzymatic function. The stability of C45 encapsulated inside an organic-solvent resistance hydrogel allowed for the development of a novel heterogeneous biocatalyst that remains catalytically active towards peroxidase and carbene transferase chemistry when suspended in a range of neat organics solvents. Overall, a de novo c-type heme-containing protein proficient at i) forming a number of stable, spectroscopically distinctive bioorganometallic intermediates, ii) catalysing a wide range of abiological chemical reactions via a etallocarbenoid intermediate, and iii) performing biocatalytic conversions under conditions typically inhospitable to solvents (i.e. neat organic solvents) is reported.
Supervisor: Anderson, Ross ; Seddon, Annela Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available