The Open Protein Structure Annotation Network
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    Table of contents
    1. 1. Protein Summary
    2. 2. Ligand Summary

    Title Crystal structure of BluB-like flavoprotein (YP_001089088.1) from CLOSTRIDIUM DIFFICILE 630 at 1.74 A resolution. To be published
    Site JCSG
    PDB Id 3eo8 Target Id 391659
    Molecular Characteristics
    Source Clostridium difficile 630
    Alias Ids TPS14648,YP_001089088.1,, 104374 Molecular Weight 24757.41 Da.
    Residues 218 Isoelectric Point 6.02
    Sequence melqdtifkrqsvrkfknqdvsdedilkmikaagaapsgkniqnwhfvvikrrdlmekiadvitkkqqe ilvemdkvsvdkanrfrkfvknftlfylkapvlvlvftkvynpsgyyelelidapketidklfirnpgm qslgaaienftlsaielgygscwltsqnyaadeieavleaetgfekgeyflgamlalgvpednlkspsk kpveeictfik
      BLAST   FFAS

    Structure Determination
    Method XRAY Chains 6
    Resolution (Å) 1.74 Rfree 0.198
    Matthews' coefficent 2.27 Rfactor 0.156
    Waters 2045 Solvent Content 45.70

    Ligand Information



    Protein Summary


    Structural Highlight: The structure of MG0383AE is another example of an FMN-containing target with uncharacterized function. The structure here shows an anion bound at the active site, and the presence of this negtaively-charged anion suggests a role for this target in oxygen reduction and further suggests that this enzyme is a flavin dependent oxygenase.






    Clostridium difficile is a ubiquitous microbe found in both human and animal feces. The bacterium is especially prevalent  in in hospital environments.Under certain circumstances, this bacterium is pathogenic. Treatment with broad spectrum antibiotics can  initiate the over colonization of C. difficile in the intestinal tract. This overgrowth of C. difficile  leads to buildup of its toxins and induces unpleasant symptoms such as diarrhea, bowel inflammation, and fever. As part of our efforts to understand this medically-relevant bacterium, we are examining the structures of proteins coded for by this organism. Here we describe the three-dimensional structure of Target ID 391559 encoded by Clostridium difficile. The structure was determined by multiwavelength anomalous dispersion from a monoclinic crystal formmonomer_ribbons.png, P2(1), to a resolution of 1.74 Angstroms.  The structure reveals that this target belongs to the alpha + beta class and belongs to the FMN-dependent nitroreductase like fold. Shown to the left is a ribbon representation of the structure of 391559, color coded with the N-terminal end in blue and the C-terminal end in red. The core of each monomer consists of a central four stranded beta-sheet surrounded on three sides by alpha-helicies. The most prominent structural feature is an FMN cofactor (violet in the figure to the left) that is situated at one end of the beta sheet. The C-terminal end of the protein (red) is positioned away from the core of the monomer to form contacts with a second monomer within a dimers.








          beta turn       gamma turn       beta hairpin
    Residue contacts:   
          to ligand       to metal




    The secondary structural elements of Target ID 391559 superimposed on the amino acid sequence.














     The biologically significant oligomerization state in solution of Target ID 391559 is the dimer. Shown to the left is a ribbon representation of the structure with the individual monomers shown in green and blue. The bound FMN cofactors are shown in red. The monomers are related by a two-fold non crystallographic dyad axis. The C-terminal end of each monomer contributes a beta strand to a symmetry-related monomer in the dimer to form a five-stranded beta sheet. In addition, each FMN cofactor interacts with residues from both monomers within the dimer.

    Target ID 391559 belongs to the PF00881 Pfam group, and this Pfam group is comprised of FMN or FAD dependent NAD(P)H dependent enzymes capable of metabolizing nitro-substituted compunds. The Protein Data Bank currently contains 71 structure that belong to this Pfam classification. Howver, even though this Pfam class shows extensive structural coverage, many of the structurally-characterized proteins in this Pfam group lack extensive functional annotation. The EBI Secondary Structure Matching program shows several proteins that have a similar fold to that of 391559.


     PDB Entry
     RMSD (Angstrom)
     Sequence Identity (%)
     1vwq  1.99  26.1 Crystal structure of a nitroreductase family protein from bacillus cereus atcc 14579
     1nox  1.70  24.5 Nadh oxidase from thermus thermophilus
     2b67  1.54  24.4

    Crystal structure of the nitroreductase family protein from streptococcus pneumoniae tigr4
     3bem  1.73  20.8 Crystal structure of putative nitroreductase ydfn (2632848) from bacillus subtilis at 1.65 a resolution
     2ifa  2.40  19.5 Crystal structure of the putative nitroreductase (smu.260) in complex with fmn from streptococcus mutans, northeast structural genomics target smr5.
     3bm1  2.16  29.4

    Crystal structure of a minimal nitroreductase ydja from escherichia coli k12 with and without fmn cofactor
     2i7h  2.17  23.0 Crystal structure of the nitroreductase-like family protein from bacillus cereus
     2hay  2.09  23.5

    The crystal structure of the putative NAD(p)h-flavin oxidoreductase from streptococcus pyogenes m1 gas
     2fre  2.18  28.0

    The crystal structure of the oxidoreductase containing fmn
     2r01  1.71  25.6 Crystal structure of putative fmn-dependent nitroreductase (np_661249.1) from chlorobium tepidum tls at 1.15 a resolution

    1NOX_to_MG0383AE.pngSuperposition of the polypeptide backbones of 391559(green) with PDB ID 1nox (blue). 1nox is the NADH oxidase from the thermophilic bacterium Thermus thermophilus. 1nox also contains bound FMN cofactors that are located at the same relative position as those in 391559. In general, the overlap shows that the regions of the two proteins surrounding FMN prosthetic groups overlap favorably, the regions of the two proteins situated further away from the FMN do not overlap as favorably.










    Electrostatic potential (red=-1kT, blue=+1kT) superimposed on a solvent accessible surface of 391559. The FMN cofacter (yellow) is situated in a channel whose openings are indicated by arrows. The channel is surrounded by a ring of postive potential.








    Ligand Summary

    MG0383AE_ligands.png Each monomer in the 391559 dimer contains an active site situated in a deep cavity shown on the electrostatic surface representation above. Three ligands are situated within this binding cavity. The figure to the right shows a ribbon representation of the dimer surrounding one of the active sites. The two monomers are shown individually in green and blue. A bound FMN cofactor is shown in red. Adjacent to and facing parallel to the FMN ring surface is a molecule of acetate  (magenta)from the crystallization solution. The electron density maps also strongly suggests that a chloride anion (orange sphere) is bound within the active site pocket.





    ligand_environmrnt.bmpShown to the left is a ribbon representation of the residues surrounding one of the active sites in the 391559 dimer. The active site is situated at the interface between the two monomers, and both subunits in the dimer make contact with the bound FMN and chloride ion. Most of the protein-ligand interactions for the FMN involve one of the monomers. (shown as green in the figure to the left). However, some residues on the second dimer (shown in blue )also make contact. A loop on the second monomer between residues 30-40 is involved in short range interaction with the FMN. In this particular instance,  the bound acetate is situtaed within hydrogen bonding distance of an oxygen atom on the FMN tail and the amide backbone nitrogen of Lys 40. The ring of the FMN is situated 3.5 Angstroms from the acetate. The postively-charged sidechains of Arg 14 on one subunit and Lys 40 on the second subunit are in the vicinity of of the bound acetate and are likely to be involved in stabilizing its binding.

          The proposed chloride ion sits deeper in the FMN binding pocket and is approximately 4.5 Angstroms from both the FMN ring and the bound acetate. During the initial stages of the crystallographic refinement, electron density at the chloride position was modeled as a water. However, to account for the magnitude of electron density at this position, a heavier element than oxygen, such as a chloride ion needed to be modeled. The chloride ion is positioned to bridge the two monomer subunits and is within interaction distance of the sidechain atoms and mainchain peptide nitrogen atom of Thr 163 on one subunit and interacts similarily with Ser 113 on the second subunit. An initial review of the literature suggests that the binding of anions such as chloride is functionally significant. The structure of vanillyl-alcohol oxisase, an FAD-containing enzyme from Penicillium simplicissimum was also shown to contain a bound chloride near the FMN ring and this binding site might represent the oxygen reduction site. The presence of a similarily positioned chloride anion in the structure described here suggests that 391559 could also function as an oxygenase.

         Ligand Binding template analysis in Profunc reveals a "Certain Match" (Profunc terminology) with the structure corresponding to 2isk. The results of this analysis shows that 18 atoms in three residues from 2isk (Arg 10E, Pro 37F, and Ser 140F) overlap with cooresonding atoms (Arg 30A, Pro B58, and Ser 144B) with an rmsd of 0.58 Angstroms. This structual homolg (PDB ID 2isk) is the protein encoded by the BluB gene from Sinorhizobium meliloti, and catalyzes the fragmentation of FMN to form a ligand of vitamin B12, 5,6-dimethylbenzimidazole and erythrose-4-phosphate. In the structure determination of the reduced form of this enzyme, a strong electron density peak, attributed to bound oxygen,  appears over the re-face of reduced flavin, rhe oxidized structure. The position is similar to that of the acetate anion proposed for the structure described here.




    Joosten, V. and van Berkel, Willem JH (2007) "Flavoenzymes"  Current Opinion in Chemical Biology Vol 11, pp. 195-202.


    Mattevi, A. (2006) "To be or not to be an oxidase:challenging the oxygen reactivity of flavoenzymes" (2006) Trends in Biochemical Sciences Vol 31, No 5


    Taga, M.E., Larsen, N.A., Howard-Jones, N.R., Walsh, C.T., and Walker, G.C. " BluB cannibalized flavin to form the lower ligand of vitamin B12" (2007) Nature vol 446 22 March 2007.





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