Investigation on the selectivity and activity of bacterial phenol coupling cytochrome P450 enzymes

Abstract: Oxidative phenol coupling is a crucial step in the biosynthesis of many biarylic secondary metabolites. The radical mechanism allows a high degree of structural diversity, in which different types of selectivity can be distinguished: site- or regioselectivity, various subtypes of stereoselectivity, chemoselectivity, and substrate selectivity in an enzymatic context. While in organic chemistry the coupling’s selectivity is challenging to control, many biocatalytic phenol coupling systems have been characterized with remarkable selectivity. Laccases and cytochrome P450 enzymes (CYPs) are the two major enzyme families that have been described for catalyzing coupling reactions. Thereby, the phenol coupling enzymes often show self-sufficient selectivity control; however, dependencies of e.g., auxiliary proteins or conditions on the selectivity were reported in certain systems.
In this work, the selectivity and activity of phenol coupling CYPs from Streptomyces spp. were investigated. Based on Präg’s model system in 2013, further biosynthetic gene clusters (BGCs) and homologous candidates also with putative different regioselectivity were discovered. For the characterization of the new candidates, the putative native substrate julichrome Q10, a key intermediate in lincolnenin and julichrome biosynthesis, was isolated from a crossover mutant, which derived from the gene inactivation process of the CYP gene linI in S. lincolnensis. In addition, monomeric julichrome derivatives, unsubstituted or methylated, were synthesized via tandem MICHAEL-DIECKMANN condensation. The derivative hydroxylated at C-3 was not available by this approach due to stability issues of the MICHAEL-acceptor unit.
For the reconstitution of the in vitro activity of the new candidates, assay conditions were screened by means of the 7,7’-selective CYP JulI with the surrogate redox partners (CamAB) from Pseudomonas putida DSM 50198. Combining cell-free extracts (CFEs) containing the three components provided the most stable results that were applied for the other candidates. However, no catalytic activity for SetI, LinI, or their homologues was detected with the tested compounds. As the corresponding BGCs revealed specific ferredoxin (Fdx) genes, an incompatibility of the surrogate reductase system CamAB was postulated. Even though, both Fdxs LinM and SetM enabled weak catalytic activity of JulI, the activity of other candidates was still elusive. To improve the catalytic efficiency, potentially suitable Fdx reductase candidates from S. lincolnensis were tested with SetM and LinM. Thereby, one promising candidate was identified that, despite poor solubility, improved the conversion to julichrome Q6-6 when combined with LinM and JulI. Notably, JulI showed self-sufficient
and stringent regioselectivity independent of the ferredoxin-type or assay conditions, which is potentially not applicable for SetI, LinI and homologues.
Despite the high amino acid sequence identities between the candidates of the different subgroups, those candidates are putatively responsible for different regioselectivities, which consequently suggests a sequence-encoded selectivity. However, an alteration of the regioselectivity of the 7,7’-selective JulI was not accomplished by mutagenesis experiments in its putative substrate binding site. Surprisingly, despite the replacement of regions containing several amino acids, a few enzyme variants still showed catalytic activity while keeping stringent 7,7’-regioselectivity. Furthermore, a self-sufficient fusion protein consisting of JulI, CamA, and CamB was generated in catalytically active form, again with 7,7’-selectivity. Prospectively, the elucidation of its structure could give more insight into the conformational changes during the catalytic mechanism, also with respect to the impact of the redox partners

Object Details

• Location: Deutsche Nationalbibliothek Frankfurt am Main
• Extent: Online-Ressource
• Language: Englisch
• Notes: Universität Freiburg, Dissertation, 2023

Classification and Topics

• Keyword: Cytochrom P-450 ; Enzymkatalyse ; Bakterien

Contributors, Places and Time

• Event: Veröffentlichung
• (where): Freiburg
• (who): Universität
• (when): 2024
• Creator: Eisen, Frederic
• Contributor: Müller, Michael ; Bechthold, Andreas ; Hoffmeister, Dirk ; Albert-Ludwigs-Universität Freiburg. Institut für Pharmazeutische Wissenschaften ; Albert-Ludwigs-Universität Freiburg. Fakultät für Chemie und Pharmazie

Further information

• URN: urn:nbn:de:bsz:25-freidok-2513653
• Rights: Open Access; Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Last update: 15.08.2025, 7:24 AM CEST