The Stereoselective Oxidation of para-Substituted Benzenes by a Cytochrome P450 Biocatalyst.

The serine 244 to aspartate (S244D) variant of the cytochrome P450 enzyme CYP199A4 was used to expand its substrate range beyond benzoic acids. Substrates, in which the carboxylate group of the benzoic acid moiety is replaced were oxidised with high activity by the S244D mutant (product formation rates >60 nmol.(nmol-CYP)-1 .min-1 ) and with total turnover numbers of up to 20,000. Ethyl α-hydroxylation was more rapid than methyl oxidation, styrene epoxidation and S-oxidation. The S244D mutant catalysed the ethyl hydroxylation, epoxidation and sulfoxidation reactions with an excess of one stereoisomer (in some instances up to >98 %). The crystal structure of 4-methoxybenzoic acid-bound CYP199A4 S244D showed that the active site architecture and the substrate orientation were similar to that of the WT enzyme. Overall, this work demonstrates that CYP199A4 can catalyse the stereoselective hydroxylation, epoxidation or sulfoxidation of substituted benzene substrates under mild conditions resulting in more sustainable transformations using this heme monooxygenase enzyme.

Analysis and preliminary characterisation of the cytochrome P450 monooxygenases from Frankia sp. EuI1c (Frankia inefficax sp.).

Frankia bacteria are nitrogen fixing species from the Actinobacterium phylum which live on the root nodules of plants. They have been hypothesised to have significant potential for natural product biosynthesis. The cytochrome P450 monooxygenase complement of Frankia sp. EuI1c (Frankia inefficax sp.), which comprises 68 members, was analysed. Several members belonged to previously uncharacterised bacterial P450 families. There was an unusually high number of CYP189 family members (21) suggesting that this family has undergone gene duplication events which are classified as "blooms". The likely electron transfer partners for the P450 enzymes were also identified and analysed. These consisted of predominantly [3Fe-4S] cluster containing ferredoxins (eight), a single [2Fe-2S] ferredoxin and a couple of ferredoxin reductases. Three of these CYP family members were produced and purified, using Escherichia coli as a host, and their substrate range was characterised. CYP1027H1 and CYP150A20 bound a broad range of norisoprenoids and terpenoids. CYP1074A2 was highly specific for certain steroids including testosterone, progesterone, stanolone and 4-androstene-3,17-dione. It is likely that steroids are the physiological substrates of CYP1074A2. These results also give an indication that terpenoids are the likely substrates of CYP1027H1 and CYP150A2. The large number of P450s belonging to distinct families as well as the associated electron transfer partners found in different Frankia strains highlights the importance of this family of enzymes has in the secondary metabolism of these bacteria.