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dc.contributor.advisorBell, Stephen G.-
dc.contributor.advisorPyke, Simon-
dc.contributor.authorSarkar, Md Raihan-
dc.description.abstractThe cytochrome P450 enzymes CYP101B1 and CYP101C1, which are from the aromatic hydrocarbon degrading bacterium Novosphingobium aromaticivorans DSM12444, can hydroxylate norisoprenoids with high activity and selectivity. With the aim of further understanding their substrate range, a selection of cyclic alkanes, ketones and alcohols were studied. Cycloalkanes were oxidised, but both enzymes displayed low binding affinity and productive activity. The presence of a ketone moiety in the cycloalkane skeleton significantly improved the substrate binding affinity and the oxidation activity. CYP101C1 catalysed the oxidation of the cycloalkanones at the C-2 position with high regioselectivity. The regioselectivity of CYP101B1 was different. It oxidised cycloalkanones at positions remote from the carbonyl group. This indicated that the binding orientation of the cyclic ketones in the active site of each enzyme must be different. Cyclic alcohols and cyclohexylacetic acid showed little to no activity with either enzyme. The introduction of an ester protecting group to these substrates significantly enhanced the monooxygenase activity. These substrates were oxidised regioselectively on the opposite side of the ring system to the ester directing group. For example, both enzymes preferentially oxidised the C-H bond at the C4, C5 and C7 position of the cyclohexyl, cyclooctyl and cyclododecyl ester compounds, respectively. In addition, certain linear ketones and esters were also found to be suitable substrates for these biocatalysts. CYP101B1 mediated metabolism of the tricyclic compounds adamantane, 1‐ and 2‐ adamantanol and 2‐adamantanone proceeds with low oxidation activity and multiple metabolites were identified. Insertion of a directing group (acetate/isobutyrate) at the alcohol of these adamantanols significantly increased the affinity, activity and coupling efficiency (productive use of reducing equivalents) of CYP101B1 compared to the parent compounds. This substrate engineering approach with these adamantyl derivatives led to a 65 to 122-fold higher product formation activity. The turnovers were also regioselective and in some instances stereoselective. Additionally, the amide N‐(1‐adamantyl)acetamide was oxidised efficiently by CYP101B1, whereas 1‐adamantylamine was not. Whole-cell biotransformation systems were used to generate the metabolites in good yield (g/L scale). Overall, the use of ester directing groups and the modification of the amine to an amide enabled CYP101B1 to oxidise the adamantane skeleton more efficiently and selectively. Wild-type (WT) CYP101B1 can catalyse the oxidation of aromatic substrates such as alkylbenzenes, alkylnaphthalenes and acenaphthene, but the binding affinities and the oxidation activities were low. Both the binding affinity and product formation activity of this enzyme for these hydrophobic substrates were enhanced using site-directed mutagenesis. The Histidine 85 (H85) of CYP101B1 aligns with tyrosine 96 of CYP101A1 (P450cam), which, in the latter enzyme forms the only hydrophilic interaction with its natural substrate, camphor. The H85 residue of CYP101B1 was therefore replaced with phenylalanine (F), and this H85F variant exhibited greater affinity and activity towards hydrophobic substrates. For instance, the product formation activity of the H85F variant for acenaphthene oxidation was increased sixfold to 245 nmol.nmol-CYP–1.min–1. This indicated that this residue is in the substrate binding pocket or the access channel of the enzyme. Methylcubanes have been used as mechanistic probes to differentiate between radical and cationic pathways in cytochrome P450 oxidation. A series of methylcubanes were designed which would place the methyl group close to reactive heme iron centre of CYP101B1. CYP101B1 efficiently oxidised the substituted methylcubane derivatives yielding the equivalent cubylmethanol in 93 ± 7 % yield. The cube was found to be intact in all the turnover products, and no methylcubanols or any other rearranged metabolites containing homocubyl were detected. These results were consistent with a rapid radical rebound step in these oxidations and argued against the involvement of any carbocation-based intermediates during the oxidation. The CYP101B1 system, which also combines a FAD-containing ferredoxin reductase and a [2Fe-2S] ferredoxin, was investigated with oxygenated aromatics including naphthols, naphthoquinones, dihydroxynaphthalene and phenols. In vitro NADH oxidation rates in both the presence and absence of CYP101B1 were fast with these substrates (≥800 min-1). Minimal metabolite formation was detected, and the majority of reducing equivalents were transformed into hydrogen peroxide. Large amount of H2O2 in these reactions in the absence of P450 indicated that the ferredoxin (Arx) and ferredoxin reductase (ArR) catalysed futile redox cycling with naphthoquinones giving rise to the uncoupling of the reducing equivalents. Further examination of naphthols and naphthoquinones together with 2-adamantyl acetate in the fully reconstituted CYP101B1 turnovers demonstrated that the presence of naphthoquinones led to diminished product formation as they interfere with the electron transfer process. This type of uncoupling in the bacterial P450 electron transfer partners containing ferredoxin system would be considered an additional form of uncoupling over those which arise in the P450 active site.en
dc.subjectCytochrome P45Osen
dc.subjectC-H bonds functionalisationen
dc.subjectsubstrate engineeringen
dc.subjectfutile redox cyclingen
dc.titleApplication of the Monooxygenase Enzymes CYP101B1 and CYP101C1 from Novosphingobium aromaticivorans for Selective and Efficient Functionalisation of Inert C-H bondsen
dc.contributor.schoolSchool of Physical Sciences : Chemistryen
dc.provenanceThis 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:
dc.description.dissertationThesis (Ph.D.) -- University of Adelaide, School of Physical Sciences, 2019en
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