TY - JOUR

T1 - Enzyme cascades for in vitro and in vivo FMN prenylation and UbiD (de)carboxylase activation under aerobic conditions

AU - Khusnutdinova, Anna N

AU - Batyrova, Khorcheska A

AU - Wang, Po-Hsiang

AU - Flick, Robert

AU - Edwards, Elizabeth A

AU - Yakunin, Alexander F

N1 - Copyright © 2024. Published by Elsevier Inc.

PY - 2024/10/16

Y1 - 2024/10/16

N2 - Microbial carboxylases and decarboxylases play important roles in the global carbon cycle and have many potential applications in biocatalysis and synthetic biology. The widespread family of reversible UbiD-like (de)carboxylases are of particular interest because these enzymes are active against a diverse range of substrates. Several characterized UbiD enzymes have been shown to catalyze reversible (de)carboxylation of aromatic and aliphatic substrates using the recently discovered prenylated FMN (prFMN) cofactor, which is produced by the associated family of UbiX FMN prenyltransferases. However, discovery and investigation of novel UbiD (de)carboxylases are delayed by our limited knowledge and the experimental complexities associated with FMN prenylation and UbiD activation resulting in the production of inactive recombinant UbiD enzymes. Therefore, there is a need for developing robust methods for efficient in vitro and in vivo FMN prenylation and UbiD activation for heterologous production of active UbiD enzymes. In this chapter, we present two protocols for in vitro and in vivo FMN prenylation and UbiD activation under aerobic conditions using enzyme cascades with regenerating systems and recombinant E. coli cells.

AB - Microbial carboxylases and decarboxylases play important roles in the global carbon cycle and have many potential applications in biocatalysis and synthetic biology. The widespread family of reversible UbiD-like (de)carboxylases are of particular interest because these enzymes are active against a diverse range of substrates. Several characterized UbiD enzymes have been shown to catalyze reversible (de)carboxylation of aromatic and aliphatic substrates using the recently discovered prenylated FMN (prFMN) cofactor, which is produced by the associated family of UbiX FMN prenyltransferases. However, discovery and investigation of novel UbiD (de)carboxylases are delayed by our limited knowledge and the experimental complexities associated with FMN prenylation and UbiD activation resulting in the production of inactive recombinant UbiD enzymes. Therefore, there is a need for developing robust methods for efficient in vitro and in vivo FMN prenylation and UbiD activation for heterologous production of active UbiD enzymes. In this chapter, we present two protocols for in vitro and in vivo FMN prenylation and UbiD activation under aerobic conditions using enzyme cascades with regenerating systems and recombinant E. coli cells.

KW - Flavin Mononucleotide/metabolism

KW - Prenylation

KW - Escherichia coli/genetics

KW - Carboxy-Lyases/metabolism

KW - Dimethylallyltranstransferase/metabolism

KW - Aerobiosis

KW - Recombinant Proteins/metabolism

KW - Enzyme Activation

KW - Enzyme Assays/methods

KW - Protein Prenylation

U2 - 10.1016/bs.mie.2024.10.015

DO - 10.1016/bs.mie.2024.10.015

M3 - Article

C2 - 39572138

VL - 708

SP - 151

EP - 173

JO - Methods in Enzymology

JF - Methods in Enzymology

SN - 0076-6879

ER -