Carbon-halogen bond formation in enzymes

Rugg, Graham (2016) Carbon-halogen bond formation in enzymes. PhD thesis, University of Glasgow.

Abstract

SyrB2, a non-haem Fe halogenase first discovered in 2005, carries out a cryptic chlorination during the biosynthesis of syringomycin E in the bacterium Pseudomonas syringae. SyrB2 chlorinates its native substrate, l-Threonine (Thr), at an unactivated methyl group. It is able to activate this highly unreactive position using an oxoferryl intermediate of its active site complex, which abstracts a hydrogen from the substrate methyl group to form a bioradical. Whilst a provisional mechanism was quickly derived from the mechanisms of similar non-haem Fe enzymes, two features of this mechanism remain unclear: firstly, the structure or structures of the oxoferryl intermediate of its active-site complex, and secondly, why SyrB2 does not hydroxylate Thr in what would appear to be a plausible side-reaction. This latter problem is believed to be the result of substrate placement, as in reaction with two non-native substrates, α-aminobutyrate (Aba) and norvaline (Nva), SyrB2 is able to function as a hydroxylase.
This thesis sets out to answer these two questions, as well as to pursue several preliminary goals. Firstly, a method validation study was carried out on several oxoferryl model complexes, which showed that B3LYP reproduced several parameters from CASPT2 benchmarks from the literature better than other tested functionals. Next, protein-substrate interactions were studied through docking and molecular dynamics simulations, which uncovered a new position for Thr. Finally, the mechanism of SyrB2 in reaction with these three substrates was investigated in a QM/MM study, which identified two likely structures of the oxoferryl active-site complex, as well as a new species in which the substrate radical intermediate coordinates to the iron complex.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Supported by funding from the Engineering and Physical Sciences Research Council.
• Keywords: Computational chemistry, SyrB2
• Subjects: Q Science > QD Chemistry
• Colleges/Schools: College of Science and Engineering > School of Chemistry
• Supervisor's Name: Senn, Dr. Hans Martin
• Date of Award: 2016
• Depositing User: Mr Graham Rugg
• Unique ID: glathesis:2016-7045
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 25 Jan 2016 13:58
• Last Modified: 17 Feb 2016 09:00
• URI: https://theses.gla.ac.uk/id/eprint/7045