Abstract
Background Ruminococcus flavefaciens is one of the predominant fiber-degrading bacteria found in the rumen of herbivores. Bioinformatic analysis of the recently sequenced genome indicated that this bacterium produces one of the most intricate cellulosome systems known to date. A distinct ORF, encoding for a multi-modular protein, RflaF_05439, was discovered during mining of the genome sequence. It is composed of two tandem modules of currently undefined function that share 45% identity and a C-terminal X-dockerin modular dyad. Gaining insight into the diversity, architecture and organization of different types of proteins in the cellulosome system is essential for broadening our understanding of a multi-enzyme complex, considered to be one of the most efficient systems for plant cell wall polysaccharide degradation in nature.Methodology/Principal FindingsFollowing bioinformatic analysis, the second tandem module of RflaF_05439 was cloned and its selenium-labeled derivative was expressed and crystallized. The crystals belong to space group P21 with unit-cell parameters of a = 65.81, b = 60.61, c = 66.13 Å, β = 107.66° and contain two protein molecules in the asymmetric unit. The crystal structure was determined at 1.38-Å resolution by X-ray diffraction using the single-wavelength anomalous dispersion (SAD) method and was refined to Rfactor and Rfree of 0.127 and 0.152 respectively. The protein molecule mainly comprises a β-sheet flanked by short α-helixes, and a globular α-helical domain. The structure was found to be structurally similar to members of the NlpC/P60 superfamily of cysteine peptidases.Conclusions/SignificanceThe 3D structure of the second repeat of the RflaF_05439 enabled us to propose a role for the currently undefined function of this protein. Its putative function as a cysteine peptidase is inferred from in silico structural homology studies. It is therefore apparent that cellulosomes integrate proteins with other functions in addition to the classic well-defined carbohydrate active enzymes.
Highlights
Plant cell wall polysaccharides offer an extraordinary source of carbon and energy that can be utilized by various microorganisms, contributing a central component to the carbon cycle
Cellulose is the main structural component of the plant cell wall. It is arranged in highly recalcitrant fibrils which are usually embedded in a colloidal matrix of hemicellulose and lignin [1]
The cellulolytic, fiber-degrading bacterium Ruminococcus flavefaciens is one of the critically important inhabitants in the rumen of herbivores, which plays a central role in the degradation of plant cell wall fiber
Summary
Plant cell wall polysaccharides offer an extraordinary source of carbon and energy that can be utilized by various microorganisms, contributing a central component to the carbon cycle. The parent protein, RflaF_05439 (accession number: ZP_06142651), consists of a tandem repeat of two conserved modules (Figure 2) at the N terminus and a C-terminal XDoc, which binds to ScaE-Coh (unpublished results). In silico analysis of the structure implied that the protein belongs to an established superfamily of cysteine-like peptidases, termed NlpC/P60, and this domain will hereafter be coined NlpC/P60_2.
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