The prFMNH2-binding chaperone LpdD assists UbiD decarboxylase activation

Abstract

The UbiD enzyme family of prenylated flavin (prFMN)-dependent reversible decarboxylases is near ubiquitously present in microbes. For some UbiD family members, enzyme activation through prFMNH2-binding and subsequent oxidative maturation of the cofactor readily occurs, both in vivo in a heterologous host and through in vitro reconstitution However, isolation of the active holo-enzyme has proven intractable for others, notably the canonical E. coli UbiD. We show E. coli heterologous expression of the small protein LpdD - associated with the UbiD-like gallate decarboxylase LpdC from Lactobacillus plantarum - unexpectedly leads to 3,4-dihydroxybenzoic acid decarboxylation whole cell activity. This activity was shown to be linked to endogenous E. coli ubiD expression levels. The crystal structure of the purified LpdD reveals a dimeric protein with structural similarity to the eukaryotic heterodimeric proteasome assembly chaperone Pba3/4. Solution studies demonstrate LpdD protein specifically binds to reduced prFMN species only. The addition of the LpdD:prFMNH2 complex supports reconstitution and activation of the purified E. coli apo-UbiD in vitro, leading to modest 3,4-dihydroxybenzoic acid decarboxylation. These observations suggest LpdD acts as a prFMNH2-binding chaperone, enabling apo-UbiD activation through enhanced prFMNH2 incorporation and subsequent oxidative maturation. Hence, while a single highly conserved flavin prenyltransferase UbiX is found associated with UbiD enzymes, our observations suggest considerable diversity in UbiD maturation, ranging from robust autocatalytic to chaperone-mediated processes. Unlocking the full (de)carboxylation scope of the UbiD-enzyme family will thus require more than UbiX co-expression.