Please use this identifier to cite or link to this item:
Scopus Web of ScienceĀ® Altmetric
Type: Theses
Title: Isolation of new P450s and the modification of existing P450s for biocatalysis
Author: Lau, Ian
Issue Date: 2017
School/Discipline: School of Physical Sciences
Abstract: Cytochrome P450s are a family of heme-containing monooxygenases that are ubiquitous in nature. Many P450s from bacterial sources, such as from Frankia sp. EuI1c which contain genes encoding 68 of these enzymes, have not previously been investigated. These P450s are potentially involved in the metabolism and biosynthesis of novel natural products including steroids, siderophores, fatty acids and antibiotics. Here four of these P450s were successfully expressed and purified. One of these, FraEu2494, was purified at high concentrations suitable for crystallisation. A selection of chemical compounds was screened with these enzymes to determine the substrate range of these P450s. In particular the P450 FraEu1415 exhibited high affinity towards steroid compounds such as testosterone, estrone and progesterone, highlighting the important compounds these P450s may metabolise. The ferredoxin electron transfer partners of these P450s were also successfully expressed and purified. While the ferredoxin reductases were unable to be produced, a mutant library of the ferredoxin Fdx2495 was created. Non-standard amino acid residues within the iron-sulfur binding motif of the ferredoxins were investigated to ascertain if Fdx2495 could be used as a model for future study. The cytochrome P450 CYP199A4 from Rhodopseudomonas palustris strain HaA2 is highly specific for the regioselective oxidation of para-substituted benzoic acids such as 4-methoxybenzoic acid. It has been reported that the activity of the CYP199A4 S244D mutant for the hydroxylation and demethylation of para-substituted non-benzoic acid derivatives is greater than with the wild-type enzyme. Here we report the potential scale up of these oxidation reactions by the S244D mutant with a system that contains an excess of NADH. A selection of similar para-substituted compounds, including styrenes, methylthio- and dimethyl-substituted benzene derivatives were tested with the enzyme to further investigate the mechanism and productivity of the mutant. The sulfoxidation and epoxidation reactions by the S244D mutant of para-substituted benzene derivatives were investigated. The epoxidation reactions produced small amounts of aldehyde arising from a 1,2-rearrangement reaction, giving evidence of a non-concerted reaction pathway. Chiral analysis for the sulfoxidation and epoxidation reactions revealed a consistent bias for a single enantiomer, suggesting similar binding conformations for these para-substituted benzene substrates within the active site. The crystal structure of 4-methoxybenzoic acid bound to the CYP199A4 S244D mutant (PDB: 5U5J) was solved. This revealed small differences between the mutant and the equivalent wild-type structure (PDB: 4DO1). Aside from the specific 244 amino acid mutation, the substrate binding site was largely unaffected by the S244D variant. A small shift in the position of the substrate over the active site was observed. A large shift was discovered for the chloride ion which caps the active site from external solvent in the wild-type enzyme. The chloride ion in the S244D mutant coordinates to an asparagine residue, which plays no role in the coordination to this ion in the equivalent wild-type.
Advisor: Bell, Stephen G.
Harris, Hugh Hamlyn
Dissertation Note: Thesis (M.Phil.) -- University of Adelaide, School of Physical Sciences, 2017.
Keywords: chemistry
cytochrome P450
Provenance: This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at:
DOI: 10.4225/55/5a1e336d31769
Appears in Collections:Research Theses

Files in This Item:
File Description SizeFormat 
01front.pdf168.92 kBAdobe PDFView/Open
02whole.pdf53.34 MBAdobe PDFView/Open
  Restricted Access
Library staff access only196.12 kBAdobe PDFView/Open
  Restricted Access
Library staff access only53.59 MBAdobe PDFView/Open

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.