Sequence-based Characterization of Ferredoxin-like Proteins in Eubacterium callanderi KIST612

Abstract

Acetogenic bacteria (acetogen) are the microbial groups capable of syngas fermentation and are promising biocatalysts to convert gaseous substrates into various value-added products such as acetate and butyrate. Acetogen utilize the reductive acetyl-CoA pathway to produce the building block, acetyl-CoA which is a precursor of acetate. In the reductive acetyl-CoA pathway that is also known as the Wood-Ljungdahl pathway, a series of enzymatic reactions require high reducing power to produce metabolites from CO2. Among native electron carriers, ferredoxin can achieve the energy and it is used as a major reducing agent of acetogen because it has a low redox potential. Ferredoxins are proteinaceous electron carriers containing an iron-sulfur cluster as a cofactor and they have a wide range of low redox potentials from -100 mV to -700 mV depending on the iron sulfur cluster types. That is, ferredoxins play a role as important electron carriers to facilitate thermodynamically unfavorable redox reactions such as CO2 reduction by providing reducing equivalent(s) to the reactants. Therefore, it is necessary to understand the biochemical properties of ferredoxin to improve the productivity of metabolites in acetogen. Eubacterium callanderi KIST612 is a representative acetogen and whole genome sequencing analysis and omics analysis were performed. It was confirmed that E. callanderi KIST612 has multiple genes encoding ferredoxin. However, the characteristics of individual ferredoxins remain poorly understood. Herein, nine ferredoxin candidates of E. callanderi KIST612 were characterized to analyze sequence properties and to presume protein function by performing protein structural comparative analysis. The results of Sequence Similarity Network (SSN) and the conserved motif analysis indicated that ferredoxin candidates could be classified into two groups according to [Fe-S] cluster types, [4Fe-4S] and [2Fe-2S] types. The protein folding structure models showed that [4Fe-4S] cluster motif-containing proteins which are ELI_1388 and ELI_4562 found in E. callanderi KIST612 had the highest structural similarity with a ferredoxin from Clostridium pasteurianum (RMSD = 1.208 Å and 1.084 Å, respectively), which is widely used as a reducing agent in the redox studies of acetogens. On the other hand, [2Fe-2S] cluster motif-containing proteins which are ELI_2009 and ELI_3608 found in E. callanderi KIST612 had the highest structural similarity with a reductive activator of corrinoid iron-sulfur protein from Carboxydothermus hydrogenoformans (RMSD = 0.967 Å and 0.942 Å, respectively). However, only ELI_3608 possesses [2Fe-2S] cluster binding site at the N-terminal domain and the electron transition through [2Fe-2S] cluster of ELI_3608 was validated by spectroscopic measurements. As a result, we proposed electron transport mechanisms according to the iron-sulfur cluster. It was presumed that ELI_3608 of [2Fe-2S] cluster-containing proteins and ELI_1388 and ELI_4562 of [4Fe-4S] cluster-containing proteins might play a role as plausible electron carriers for enzymatic redox reactions. Although in vivo and in vitro assays should be performed to validate the assumption, this study could contribute to understanding the genetic and structural characteristics of ferredoxin-like proteins in E. callanderi KIST612.