PLoS ONE (Jan 2013)

Crystal structure of an uncommon cellulosome-related protein module from Ruminococcus flavefaciens that resembles papain-like cysteine peptidases.

  • Maly Levy-Assaraf,
  • Milana Voronov-Goldman,
  • Inna Rozman Grinberg,
  • Gloria Weiserman,
  • Linda J W Shimon,
  • Sadanari Jindou,
  • Ilya Borovok,
  • Bryan A White,
  • Edward A Bayer,
  • Raphael Lamed,
  • Felix Frolow

DOI
https://doi.org/10.1371/journal.pone.0056138
Journal volume & issue
Vol. 8, no. 2
p. e56138

Abstract

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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.Following 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.The 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.