Die Plant Science Student Conference (PSSC) wird seit 20 Jahren im jährlichen Wechsel von Studierenden der beiden Leibniz-Institute IPK und IPB organisiert. Im Interview erläutern Christina Wäsch (IPK) und Carolin Apel (IPB),…
Über 600 Gäste kamen am 4. Juli ans IPB zur Langen Nacht, die Wissen schafft, um bei unserem Wissenschafts-Quiz-Parcours viel Neues zu erfahren und ihre Kenntnisse unter Beweis zu stellen. Unser Programm in diesem Jahr…
Herrmann, S.; Dippe, M.; Pecher, P.; Funke, E.; Pietzsch, M.; Wessjohann, L. A.;Engineered bacterial flavin‐dependent monooxygenases for the regiospecific hydroxylation of polycyclic phenolsChemBioChem23e202100480(2022)DOI: 10.1002/cbic.202100480
4-Hydroxyphenylacetate 3-hydroxylase (4HPA3H), a flavin-dependent monooxygenase from E. coli that catalyzes the hydroxylation of monophenols to catechols, was modified by rational re-design to convert also more bulky substrates, especially phenolic natural products like phenylpropanoids, flavones or coumarins. Selected amino acid positions in the binding pocket of 4HPA3H were exchanged by residues from the homologous protein from Pseudomonas aeruginosa, yielding variants with improved conversion of spacious substrates such as the flavonoid naringenin or the alkaloid mimetic 2-hydroxycarbazole. Reactions were followed by an adapted Fe(III)-catechol chromogenic assay selective for the products. Especially substitution of the residue Y301 facilitated modulation of substrate specificity: introduction of non-aromatic but hydrophobic (iso)leucine resulted in the preference of the substrate ferulic acid (having a guaiacyl (guajacyl) moiety, part of the vanilloid motif) over unsubstituted monophenols. The in vivo (whole-cell biocatalysts) and in vitro (three-enzyme cascade) transformations of substrates by 4HPA3H and its optimized variants was strictly regiospecific and proceeded without generation of by-products.
Publikation
Dippe, M.; Davari, M. D.; Weigel, B.; Heinke, R.; Vogt, T.; Wessjohann, L. A.;Altering the regiospecificity of a catechol
O‐methyltransferase through rational design: Vanilloid vs. isovanilloid motifs in the B‐ring of flavonoidsChemCatChem14e202200511(2022)DOI: 10.1002/cctc.202200511
Rational re-design of the substrate pocket of phenylpropanoid-flavonoid O-methyltransferase (PFOMT) from Mesembryanthe-mum crystallinum, an enzyme that selectively methylates the 3’-position (= meta-position) in catechol-moieties of flavonoids to guiacol-moieties, provided the basis for the generation of variants with opposite, i. e. 4’- (para-) regioselectivity and enhanced catalytic efficiency. A double variant (Y51R/N202W) identified through a newly developed colorimetric assay efficiently modified the para-position in flavanone and flavano-nol substrates, providing access to the sweetener molecule hesperetin and other rare plant flavonoids having an isovanil-loid motif.