Site-selective chlorination of pyrrolic heterocycles by flavin dependent enzyme PrnC.

Halogenation of pyrrole requires strong electrophilic reagents and often leads to undesired polyhalogenated products. Biocatalytic halogenation is a highly attractive approach given its chemoselectivity and benign reaction conditions. While there are several reports of enzymatic phenol and indole halogenation in organic synthesis, corresponding reports on enzymatic pyrrole halogenation have been lacking. Here we describe the in vitro functional and structural characterization of PrnC, a flavin-dependent halogenase that can act on free-standing pyrroles. Computational modeling and site mutagenesis studies identified three key residues in the catalytic pocket. A moderate resolution map using single-particle cryogenic electron microscopy reveals PrnC to be a dimer. This native PrnC can halogenate a library of structurally diverse pyrrolic heterocycles in a site-selective manner and be applied in the chemoenzymatic synthesis of a chlorinated analog of the agrochemical fungicide Fludioxonil.

Expression and engineering of PET-degrading enzymes from Microbispora, Nonomuraea, and Micromonospora.

IMPORTANCE: Mismanagement of PET plastic waste significantly threatens human and environmental health. Together with the relentless increase in plastic production, plastic pollution is an issue of rising concern. In response to this challenge, scientists are investigating eco-friendly approaches, such as bioprocessing and microbial factories, to sustainably manage the growing quantity of plastic waste in our ecosystem. Industrial applicability of enzymes capable of degrading PET is limited by numerous factors, including their scarcity in nature. The objective of this study is to enhance our understanding of this group of enzymes by identifying and characterizing novel enzymes that can facilitate the breakdown of PET waste. This data will expand the enzymatic repertoire and provide valuable insights into the prerequisites for successful PET degradation.

Direct arene trifluoromethylation enabled by promiscuous activity of fungal laccase.

Laccase from Trametes versicolor was found to oxidize non-phenolic arenes and enable the trifluoromethylation of arenes in the presence of in situ generated CF3 radicals at a catalyst loading as low as 0.0034%. The biocatalytic trifluoromethylation proceeded under mild conditions and could increase the yield by up to 12 fold, compared to the control.

Further Characterization of Fungal Halogenase RadH and Its Homologs.

RadH is one of the flavin-dependent halogenases that has previously exhibited promising catalytic activity towards hydroxycoumarin, hydroxyisoquinoline, and phenolic derivatives. Here, we evaluated new functional homologs of RadH and expanded its specificities for the halogenation of non-tryptophan-derived, heterocyclic scaffolds. Our investigation revealed that RadH could effectively halogenate hydroxyquinoline and hydroxybenzothiophene. Assay optimization studies revealed the need to balance the various co-factor concentrations and where a GDHi co-factor recycling system most significantly improves the conversion and efficiency of the reaction. A crystal structure of RadH was also obtained with a resolution of 2.4 Å, and docking studies were conducted to pinpoint the binding and catalytic sites for substrates.