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3gr3

    Table of contents
    1. 1. Protein Summary
    2. 2. Ligand Summary

    Title Crystal structure of nitroreductase-like family protein (YP_033442.1) from BARTONELLA HENSELAE HOUSTON-1 at 1.45 A resolution. To be published
    Site JCSG
    PDB Id 3gr3 Target Id 390524
    Molecular Characteristics
    Source Bartonella henselae str. houston-1
    Alias Ids TPS14579,YP_033442.1, 3.40.109.10, 88979 Molecular Weight 26225.94 Da.
    Residues 229 Isoelectric Point 8.78
    Sequence madapidifqsilsrksiraftdqpvtqetireilklaarapsgtnlqpwqvivltgkilqkvgqelsq lvlsgikgereyhyyprqwrepylsrrrkvgldlykslgiqkgdqekmlhqkaknflfygapvgllfti dhdmemgswldlgmfmqtimlaargfgldtcaqaafadyhkqirsllsvpsdrhiicgmalgyrdmnap ennfeterepidnfvhfiksyp
      BLAST   FFAS

    Structure Determination
    Method XRAY Chains 2
    Resolution (Å) 1.45 Rfree 0.170
    Matthews' coefficent 2.17 Rfactor 0.146
    Waters 567 Solvent Content 43.25

    Ligand Information
    Ligands
    Metals

    Jmol

     
    Google Scholar output for 3gr3
    1. Biological and structural characterization of the Mycobacterium smegmatis nitroreductase NfnB, and its role in benzothiazinone resistance
    G Manina, M Bellinzoni, MR Pasca - Molecular , 2010 - Wiley Online Library
     
    2. Distributed structure determination at the JCSG
    H van den Bedem, G Wolf, Q Xu - Section D: Biological , 2011 - scripts.iucr.org
     

    Protein Summary

    Gene BH06130 from Bartonella henselae strain Houston-1 encodes the YP_033442 protein that has been annotated as belonging to the nitroreductase family (PF00881).

    Pre-SCOP classifies 3gr3 in the alpha+beta class, FMN-dependent nitroreductase-like superfamily, NADH oxidase/flavin reductase family. The 3gr3 structure, solved by seleno-methionine MAD to a resolution of 1.45 Angstroms, reveals the protein to be homodimeric (also supported by static light scattering analysis). The dimer interface is extensive, contributed in part by the interactions made between the C-terminal beta strand (strand 5) of one monomer and beta strand 1 of the other monomer to form a 5-stranded beta sheet. In addition, flavin mononucleotide (FMN) cofactors are tightly bound in the highly conserved solvent-accessible clefts formed at the dimer interfaces (Figure 1).

    Figure 1.  Homodimer structure of 3gr3.  Monomers are shown in blue and orange, FMN cofactors as green sticks, and unidentified ligand (UNL) as magenta sticks.

    MG10707E_dimer_2.png

     

    A search for structurally similar proteins to 3gr3 using DALI returns as top hits the flavoprotien 3eo8 (Z=19), the putative nitroreductase 3gfa (Z=18), the nitroreductase family protein 1ywq (Z=18), the flavin nitroreductase 3gbh (Z=17) and the oxygen insensitive nitroreductase 1yki (Z=15). Both sequence (PSI-BLAST) and structural (VAST) database searches were performed, and while the top results from these searches did not overlap with one another (no structures are currently known yet for any of the top sequence homologs produced by PSI-BLAST), both searches resulted in 3gr3 being similar to proteins mostly classified as nitroreductases (Table 1).

     

    Table 1. 5 best structurally similar proteins to 3gr3 according to a VAST search. 

     

     

     

     

     PDB C D
    Ali. Len.
    SCORE
    P-VAL
    RMSD
    %Id
    Description
     
    1V5Y A 

    164

    12.7

    10e-9.1

    2.4

    12.2

    Binding Of Coumarins To Nad(P)h:fmn Oxidoreductase

     
    1YKI B 

    161

    12.9

    10e-8.6

    2.2

    19.3

    The Structure Of E. Coli Nitroreductase Bound With The Antibiotic Nitrofurazone

     
    1YWQ A 

    160

    14.0

    10e-13.2

    2.2

    19.4

    Crystal Structure Of A Nitroreductase Family Protein From Bacillus Cereus Atcc 14579

     
    3EO8 F 

    157

    14.3

    10e-10.8

    2.2

    22.9

    Crystal Structure Of Blub-Like Flavoprotein (Yp_001089088.1) From Clostridium Difficile 630 At 1.74 A Resolution

     
    2IFA B 

    156

    13.8

    10e-10.7

    2.2

    17.3

    Crystal Structure Of The Putative Nitroreductase (Smu.260) In Complex With Fmn From Streptococcus Mutans, Northeast Structural Genomics Target Smr5

     

    Superpositions of 3gr3 with these structures reveal a very similar core region but that 3gr3 differs significantly in a couple of the outer regions (Figures 2a-c). One prominent difference is that 3gr3 contains an additional ~40 residues between alpha helices 3 and 5, which take the form of a long loop and an additional alpha helix (helix 4), that are absent in the other structures. This additional segment (colored green in Figure 2b and cyan in Figure 2c), which spans approximately from G74 to Q114, is structured in such a way that it, along with alpha helices 3 and 5, form almost a square frame that surrounds the entrance of the cleft leading to the putative FMN bound active site (Figures 2b,c). Another noteworthy difference is that the segment between beta-strand 2 and alpha-helix 6 is considerably shorter in 3gr3 than in the other structures. While this region is a short loop in 3gr3 (colored red in Figure 2b), this segment in 3EO8, for example, is ~25 residues longer and folds into two alpha helices. This difference is even more pronounced when 3gr3 is compared to 1YKI, in which case the two alpha helices (colored orange in Figure 2c) in this region comprise an additional 45 residues and extend over, and thus partially occlude, the active site cleft.

     

    Figure 2. (a) The aligned sequences, with secondary structure assignments, of 3gr3 with VAST detected similar structures indicate two regions with significant structural differences. (b) Structure of a 3gr3 monomer showing the additional (green) and missing (red) regions of 3gr3 when compared to its structural neighbors. (c) Superposition of 3gr3 (blue) with 1YKI (yellow) showing the major differences (cyan and orange, respectively) between the two structures near the FMN ligand site.

     

    2(a)

    VAST_structures.png

     

    2(b)                                                                                        2(c)

    MG10707E_differences.pngMG10707E_1YKI_superimpose_2.png

    Ligand Summary

    An unidentified ligand (UNL) has been modeled into each of the two putative active sites based on the Fo-Fc and 2Fo-Fc electron density (Figure 3). The UNL is located ~3.6 Angstroms directly above the FMN isoalloxazine ring system.

     

    Some of its homologs (i.e. ZP_02189579) are annotated as  p-Nitrobenzoate reductase.

     

    Figure 3. Fo-Fc electron density (green, 3 sigma contour level) calculated with the UNL omitted, at the putative FMN active site. The UNL is located between the FMN and a highly conserved tyrosine residue (Y84).

     

    UNL_omit_map_3.png 

     

    Based on the PSI-BLAST search, residues that are highly conserved at the putative active site include R19, Y84, R96, Y104, N124, and F125. Of these residues, Y84 appears particularly important as it is located ~4 Angstroms away from the reactive N5 atom of the FMN and makes multiple interactions with the UNL (Figures 3 and 4). Several of these conserved residues, including Y84, R96, and Y104, are located on the additional loop-helix segment discussed above.

     

    Superposition of 390524 with the structures of several nitroreductases bound with the antibiotic nitrofurazone (1YKI), and the inhibitors benozoate (1KQB) and acetate (1KQC) show that these small molecules partially overlap with the UNL, further supporting the notion that the UNL is at the active site (Figure 4).

     

    Figure 4. View of putative active site upon superposition of 390524 (blue) with 1YKI (orange), 1KQB (green), and 1KQC (magenta). Small molecules nitrofurazone (orange), benzoate (green), and acetate (magenta) completely or partially overlap the electron density of the UNL. Electron density is that of 2Fo-Fc (blue, contoured at 1 sigma) and Fo-Fc (green, contoured at 3 sigmas) UNL-omit maps.

     

    UNL_inhib_superimpose_3_labeled.png 

     

    The shape of the electron density for the UNL is similar to that of citrate; however, citrate was not modeled into the structure in the end because there is a break in the electron density in the middle and also because there is an additional moiety at one of the termini that is unaccounted for by citrate.

    The electron density of the UNL suggests that the ligand may consist of some type of ring structure in the middle and groups with the shape of an amide, carboxyl or nitro moiety at the terminal regions. Fitting nitrofurazone from 1YKI into the electron density of UNL-1 was attempted, and while UNL does not appear to be nitrofurazone, based on the partial fit, UNL may be a molecule similar to it (Figure 5).

     

    Figure 5. The electron density of the UNL partially resembles nitrofurazone (magenta), suggesting that the UNL may be a similar type of molecule. Electron density is the same as in Figure 4.

     

    UNL1_NFZ_docked.png

     

     

    REFERENCES:

    Reduction of polynitroaromatic compounds: the bacterial nitroreductases.  Roldán MD, Pérez-Reinado E, Castillo F, Moreno-Vivián C. FEMS Microbiol Rev. 2008 May;32(3):474-500.

    Structural and mechanistic studies of Escherichia coli nitroreductase with the antibiotic nitrofurazone. Reversed binding orientations in different redox states of the enzyme.  Race PR, Lovering AL, Green RM, Ossor A, White SA, Searle PF, Wrighton CJ, Hyde EI.  J Biol Chem. 2005 Apr 8;280(14):13256-64.

    Structures of nitroreductase in three states: effects of inhibitor binding and reduction.  Haynes CA, Koder RL, Miller AF, Rodgers DW. J Biol Chem. 2002 Mar 29;277(13):11513-20.

    Reviews

    References

     

    No references found.

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    Files (12)

    FileSizeDateAttached by 
     MG10707E_1YKI_superimpose_2.png
    Superposition of 390524 and 1YKI
    173.9 kB07:10, 21 Feb 2009ayehActions
     MG10707E_differences.png
    390524 differences
    130.35 kB07:10, 21 Feb 2009ayehActions
     MG10707E_dimer.png
    Dimer structure of 390524
    214.85 kB19:43, 20 Feb 2009ayehActions
     MG10707E_dimer_2.png
    No description
    227.91 kB00:57, 8 Apr 2009ayehActions
     MG10707E_UNL1_UNL2_Ligplot.png
    UNL-1, UNL-2 interactions
    11.09 kB07:10, 21 Feb 2009ayehActions
     MG10707E_UNL3_Ligplot.png
    UNL-3 interactions
    9.05 kB07:10, 21 Feb 2009ayehActions
     UNL1_NFZ_docked.png
    NFZ docked into UNL-1 density
    232.15 kB07:10, 21 Feb 2009ayehActions
     UNL_inhib_superimpose_2_labeled.png
    Superposition of UNL-1 with antibiotic and inhibitors
    235.06 kB07:10, 21 Feb 2009ayehActions
     UNL_inhib_superimpose_3_labeled.png
    No description
    194.93 kB00:57, 8 Apr 2009ayehActions
     UNL_omit_map_2.png
    UNL omit map
    549.96 kB07:10, 21 Feb 2009ayehActions
     UNL_omit_map_3.png
    No description
    393.97 kB00:57, 8 Apr 2009ayehActions
     VAST_structures.png
    Sequence alignment of VAST structural homologs
    36.5 kB07:10, 21 Feb 2009ayehActions
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