The prFMNH₂-binding chaperone LpdD assists UbiD decarboxylase activation

Area of Science:

• Biochemistry
• Enzymology
• Microbial biochemistry

Background:

• The UbiD enzyme family, crucial for microbial metabolism, utilizes prenylated flavin (prFMN) cofactors.
• While some UbiD enzymes self-activate, others, like E. coli UbiD, resist activation, hindering study.
• Understanding UbiD activation is key to exploring their full metabolic potential.

Purpose of the Study:

• To investigate the activation mechanism of the recalcitrant Escherichia coli UbiD enzyme.
• To identify factors enabling the in vitro and in vivo activation of UbiD enzymes.
• To explore the diversity of cofactor maturation processes in the UbiD enzyme family.

Main Methods:

• Heterologous expression of LpdD in E. coli to assess its effect on UbiD activity.
• Purification and structural analysis (crystallography) of LpdD.
• In vitro reconstitution assays using purified LpdD, prFMNH2, and apo-UbiD.

Main Results:

• Heterologous expression of LpdD restored 3,4-dihydroxybenzoic acid decarboxylation activity in E. coli.
• LpdD, structurally similar to proteasome assembly chaperones, specifically binds reduced prFMNH2.
• The LpdD-prFMNH2 complex facilitated the in vitro activation of purified E. coli apo-UbiD.

Conclusions:

• LpdD functions as a chaperone, mediating prFMNH2 binding and oxidative maturation for UbiD activation.
• UbiD enzyme maturation exhibits diversity, including chaperone-assisted pathways beyond the known UbiX prenyltransferase.
• Coexpression of UbiX alone is insufficient for activating all UbiD family members; chaperones are also critical.