Please use this identifier to cite or link to this item: http://hdl.handle.net/2440/127956
Type: Thesis
Title: Exploring the monooxygenase activity and selectivity of two related Cytochrome P450 enzymes
Author: Ahirwar, Saurabh Kumar
Issue Date: 2020
School/Discipline: School of Physical Sciences
Abstract: The cytochrome P450 enzymes CYP101B1 and CYP101C1 from Novosphingobium aromaticivorans DSM12444 are homologues of the CYP101D1 and CYP101D2 enzymes from the same bacterium and CYP101A1 (P450cam) from Pseudomonas putida. Both enzymes can efficiently hydroxylate norisoprenoids and related substrates in combination with the same ferredoxin reductase, ArR and a [2Fe-2S] ferredoxin, Arx, electron transfer partners. Even though the physiological substrates for both the enzymes are yet to be confirmed, the crystal structure of CYP101C1 bound to β -ionone and modelled structure of CYP101B1 has been generated. The Met82 residue of CYP101C1 aligns with the His85 residue of CYP101B1. In the crystallographic structure, this Met82 residue of CYP101C1, interacts with the carbonyl group of β -ionone, which makes it an interesting site for mutation as these could potentially alter the activity and hydroxylation of norisoprenoid substrates. CYP101B1 oxidised ẞ -ionone with the highest product formation rate (1010 ± 60 min-1). The CYP101C1 enzyme oxidised β -ionol with the highest product formation rate (1130 ± 30 min-1), whereas, the M82L-CYP101C1 mutant enzyme had the highest product formation rate (790 ± 22 min-1) with α-ionone. The selectivity for hydroxylation of norisoprenoids varies between CYP101B1 and CYP101C1. The M82L mutation however, did not change the selectivity for CYP101C1. For example, both β -damascone and β -ionone were hydroxylated at the C4 position by CYP101C1 and the M82L-CYP101C1 mutant. The CYP101B1 enzyme displayed an altered selectivity and hydroxylated these substrates predominantly at C3 position. When the substrate functional group was changed from a carbonyl to an alcohol (i.e. β-ionol), the hydroxylation occurred preferentially at the C3 position with all three enzymes. By comparing the oxidation of α -, β - and δ - substituted damascones, we found that the alkene moiety present inside the cyclohexyl ring did have an effect on the selectivity of oxidation. The β - substituted substrates are oxidised only at the C3 position by all three enzymes. The β - substituted substrates are oxidised at C3 position by CYP101B1 and at C4 position by CYP101C1 and M82L-CYP101C1. The δ - substituted substrate generates the 3,4-epoxide as the major product. To further explore the substrate range of CYP101B1 and CYP101C1, various substrates including cyclic ketones and cyclic esters were assessed to see if they induce enzyme activity and binding to the enzyme. The combinations of the best enzyme / substrates were then chosen to generate the oxidation metabolites in a larger quantity using whole-cell oxidation system to enable characterisation. The oxidation of 1-decalone by CYP101B1 generated a single major metabolite along with two minor products. The major product was characterized as 6-hydroxy-1-decalone and the minor product as 7-hydroxy-1-decalone. Comparison of the 1-decalone substrate to damascones, highlight the relationship of the oxidation metabolites 6-hydroxy-1-decalone to 4-hydroxy- β -damascone and 7-hydroxy-1-decalone to 3-hydroxy- β -damascone. Oxacyclotridecan-2-one is oxidised by CYP101B1 on a carbon opposite to the carbonyl group. Along with these, muscone and cyclopentadecanone show a dissociation constant similar to β -ionone with CYP101B1. However, the spin-state shift and activity induced by both of these substrates to CYP101B1 are comparatively smaller than ẞ -ionone. p-Tolyl acetate induced a large type-I spin-state shift and a weak binding to CYP101B1. It was oxidised at the benzylic methyl group, generating 7-hydroxy-p-tolyl acetate. Similarly, dihydroactinidiolide was also oxidised at the carbon opposite to the ester group generating 6-hydroxy-dihydroactinidiolide. However, this substrate induces a very small spin-state shift and was oxidised with low activity by CYP101B1. None of the tested substrate showed a spin-state shift larger than 10% HS or increased activity with CYP101C1.
Advisor: Bell, Stephen G.
Dissertation Note: Thesis (MPhil) -- University of Adelaide, School of Physical Sciences, 2020
Keywords: Cytochrome P450
Enzyme
monooxygenase oxidation
CYP10181
CYP101C1
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: http://www.adelaide.edu.au/legals
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