TY - JOUR

T1 - Biocatalytic in Vitro and in Vivo FMN Prenylation and (De)carboxylase Activation

AU - Batyrova, Khorcheska A

AU - Khusnutdinova, Anna N

AU - Wang, Po-Hsiang

AU - Di Leo, Rosa

AU - Flick, Robert

AU - Edwards, Elizabeth A

AU - Savchenko, Alexei

AU - Yakunin, Alexander F

PY - 2020/7/17

Y1 - 2020/7/17

N2 - Reversible UbiD-like (de)carboxylases represent a large family of mostly uncharacterized enzymes, which require the recently discovered prenylated FMN (prFMN) cofactor for activity. Functional characterization of novel UbiDs is hampered by a lack of robust protocols for prFMN generation and UbiD activation. Here, we report two systems for in vitro and in vivo FMN prenylation and UbiD activation under aerobic conditions. The in vitro one-pot prFMN cascade includes five enzymes: FMN prenyltransferase (UbiX), prenol kinase, polyphosphate kinase, formate dehydrogenase, and FMN reductase, which use prenol, polyphosphate, formate, ATP, NAD+, and FMN as substrates and cofactors. Under aerobic conditions, this cascade produced prFMN from FMN with over 98% conversion and activated purified ferulic acid decarboxylase Fdc1 from Aspergillus niger and protocatechuic acid decarboxylase ENC0058 from Enterobacter cloaceae. The in vivo system for FMN prenylation and UbiD activation is based on the coexpression of Fdc1 and UbiX in Escherichia coli cells under aerobic conditions in the presence of prenol. The in vitro and in vivo FMN prenylation cascades will facilitate functional characterization of novel UbiDs and their applications.

AB - Reversible UbiD-like (de)carboxylases represent a large family of mostly uncharacterized enzymes, which require the recently discovered prenylated FMN (prFMN) cofactor for activity. Functional characterization of novel UbiDs is hampered by a lack of robust protocols for prFMN generation and UbiD activation. Here, we report two systems for in vitro and in vivo FMN prenylation and UbiD activation under aerobic conditions. The in vitro one-pot prFMN cascade includes five enzymes: FMN prenyltransferase (UbiX), prenol kinase, polyphosphate kinase, formate dehydrogenase, and FMN reductase, which use prenol, polyphosphate, formate, ATP, NAD+, and FMN as substrates and cofactors. Under aerobic conditions, this cascade produced prFMN from FMN with over 98% conversion and activated purified ferulic acid decarboxylase Fdc1 from Aspergillus niger and protocatechuic acid decarboxylase ENC0058 from Enterobacter cloaceae. The in vivo system for FMN prenylation and UbiD activation is based on the coexpression of Fdc1 and UbiX in Escherichia coli cells under aerobic conditions in the presence of prenol. The in vitro and in vivo FMN prenylation cascades will facilitate functional characterization of novel UbiDs and their applications.

KW - Bacteria/enzymology

KW - Biocatalysis

KW - Carboxy-Lyases/chemistry

KW - Dimethylallyltranstransferase/chemistry

KW - Flavin Mononucleotide/chemical synthesis

KW - Oxidoreductases/chemistry

KW - Phosphotransferases (Alcohol Group Acceptor)/chemistry

KW - Prenylation

U2 - 10.1021/acschembio.0c00136

DO - 10.1021/acschembio.0c00136

M3 - Article

C2 - 32579338

VL - 15

SP - 1874

EP - 1882

JO - ACS Chemical Biology

JF - ACS Chemical Biology

SN - 1554-8929

IS - 7

ER -