Engineered Bacterial Flavin‐Dependent Monooxygenases for the Regiospecific Hydroxylation of Polycyclic Phenols. (9th February 2022)

• Record Type: Journal Article
• Authors: Herrmann, Susann; Dippe, Martin; Pecher, Pascal; Funke, Evelyn; Pietzsch, Markus; Wessjohann, Ludger A.
• Is Part Of: Chembiochem. Volume 23:Number 6(2022)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2022-02-09
• Subjects: biocatalysis -- catalysis -- flavoprotein monooxygenases -- hydroxylation -- whole-cell biotransformation; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.202100480
• Languages: English
• ISSNs: 1439-4227

Abstract

Abstract: 4‐Hydroxyphenylacetate 3‐hydroxylase (4HPA3H), a flavin‐dependent monooxygenase from E. coli that catalyzes the hydroxylation of monophenols to catechols, was modified by rational redesign 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 with 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 nonaromatic 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 byproducts. Abstract : The ability to perform regiospecific hydroxylations of sterically challenging monophenols was engineered into a flavin‐dependent monooxygenase. Its variants are valuable tools for production of the corresponding catechols for in vitro or live whole‐cell conversions.