The Membrane-Bound C Subunit of Reductive Dehalogenases: Topology Analysis and Reconstitution of the FMN-Binding Domain of PceC.

Géraldine F Buttet,Mathilde S Willemin,Julien Maillard,Aamani Rupakula,Romain Hamelin

doi:10.3389/fmicb.2018.00755

Géraldine F Buttet, Mathilde S Willemin + Show 3 more

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https://doi.org/10.3389/fmicb.2018.00755

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Abstract

Organohalide respiration (OHR) is the energy metabolism of anaerobic bacteria able to use halogenated organic compounds as terminal electron acceptors. While the terminal enzymes in OHR, so-called reductive dehalogenases, are well-characterized, the identity of proteins potentially involved in electron transfer to the terminal enzymes remains elusive. Among the accessory genes identified in OHR gene clusters, the C subunit (rdhC) could well code for the missing redox protein between the quinol pool and the reductive dehalogenase, although it was initially proposed to act as transcriptional regulator. RdhC sequences are characterized by the presence of multiple transmembrane segments, a flavin mononucleotide (FMN) binding motif and two conserved CX3CP motifs. Based on these features, we propose a curated selection of RdhC proteins identified in general sequence databases. Beside the Firmicutes from which RdhC sequences were initially identified, the identified sequences belong to three additional phyla, the Chloroflexi, the Proteobacteria, and the Bacteriodetes. The diversity of RdhC sequences mostly respects the phylogenetic distribution, suggesting that rdhC genes emerged relatively early in the evolution of the OHR metabolism. PceC, the C subunit of the tetrachloroethene (PCE) reductive dehalogenase is encoded by the conserved pceABCT gene cluster identified in Dehalobacter restrictus PER-K23 and in several strains of Desulfitobacterium hafniense. Surfaceome analysis of D. restrictus cells confirmed the predicted topology of the FMN-binding domain (FBD) of PceC that is the exocytoplasmic face of the membrane. Starting from inclusion bodies of a recombinant FBD protein, strategies for successful assembly of the FMN cofactor and refolding were achieved with the use of the flavin-trafficking protein from D. hafniense TCE1. Mass spectrometry analysis and site-directed mutagenesis of rFBD revealed that threonine-168 of PceC is binding FMN covalently. Our results suggest that PceC, and more generally RdhC proteins, may play a role in electron transfer in the metabolism of OHR.

Highlights

Organohalide respiration (OHR) is a respiratory metabolism that uses halogenated compounds as terminal electron acceptors, and allows an increasing number of anaerobic bacteria to conserve energy (Adrian and Löffler, 2016)
While there is an extensive body of information on the reductive dehalogenases (RdhA, RDases), the key enzymes involved in the catalytic reduction of organohalides, relatively little is known about the electron transport in OHR, and about the redox proteins involved in donating electrons to RDases
While menaquinones are involved in electron transfer to RDases in D. restrictus (Schumacher and Holliger, 1996), Desulfitobacterium dehalogenans (Kruse et al, 2015) and Sulfurospirillum multivorans (Miller et al, 1996), organohaliderespiring bacteria (OHRB) belonging to the Chloroflexi (Dehalococcoides and Dehalogenimonas) do not use quinones, suggesting that different pathways are used for conserving energy via OHR (Fincker and Spormann, 2017)

Summary

Introduction

Organohalide respiration (OHR) is a respiratory metabolism that uses halogenated compounds as terminal electron acceptors, and allows an increasing number of anaerobic bacteria to conserve energy (Adrian and Löffler, 2016). Models of electron transport have been recently proposed (Goris et al, 2015b; Kublik et al, 2016; Maillard and Holliger, 2016; Fincker and Spormann, 2017), indicating that, depending on their phylogeny, organohaliderespiring bacteria (OHRB) must have developed various strategies to deliver electrons to the corrinoid cofactor of RDases at sufficiently low redox potential. While menaquinones are involved in electron transfer to RDases in D. restrictus (Schumacher and Holliger, 1996), Desulfitobacterium dehalogenans (Kruse et al, 2015) and Sulfurospirillum multivorans (Miller et al, 1996), OHRB belonging to the Chloroflexi (Dehalococcoides and Dehalogenimonas) do not use quinones, suggesting that different pathways are used for conserving energy via OHR (Fincker and Spormann, 2017).

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