ENZYMES AND PROTEINS

Zinc-Independent Folate Biosynthesis: Genetic, Biochemical, and Structural Investigations Reveal New Metal Dependence for GTP Cyclohydrolase IB

Banumathi Sankaran, Shilah A. Bonnett, Kinjal Shah, Scott Gabriel, Robert Reddy, Paul Schimmel, Dmitry A. Rodionov, Valérie de Crécy-Lagard, John D. Helmann, Dirk Iwata-Reuyl, Manal A. Swairjo
DOI: 10.1128/JB.00287-09

Article Figures & Data

Figures

  • FIG. 1.
    FIG. 1.

    Reaction catalyzed by GCYH-I, and metabolic fate of 7,8-dihydroneopterin triphosphate.

  • FIG. 2.
    FIG. 2.

    GCYH-IB-dependent complementation of the B. subtilis ΔfolE mutant strain. Growth curves in defined minimal medium for wild-type (WT) and mutant B. subtilis strains expressing either or both types of GCYH-I. Results for the wild-type strain expressing GCYH-IA (⧫), the ΔfolE2 strain expressing GCYH-IA (◊), the ΔfolE strain with delayed expression of GCYH-IB (▪), the ΔfolE Δzur strain expressing GCYH-IB (▴), and the ΔfolE2 ΔfolE Δzur strain with no GCYH-IA or IB expression (•) are shown.

  • FIG. 3.
    FIG. 3.

    B. subtilis GCYH-IB enzymatic activity versus Mn2+ (○) or Zn2+ (•). Each data point represents the average results for four sets of triplicate assays (error bars correspond to the standard deviation in the data).

  • FIG. 4.
    FIG. 4.

    HPLC gel filtration analysis of N. gonorrhoeae GCYH-IB. GCYH-IB elutes at 9.22 min. The broad trailing peak at ∼12 min is due to a small molecule contaminant (retention time is after cytochrome c). The inset graph shows the standard curve presented as log MW versus Ve /V 0 where V e is the measured elution volume and V 0 is the volume of a completed excluded solute. The protein standards β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa) are plotted as closed squares (▪). The N. gonorrhoeae GCYH-IB data are plotted as open squares (□). Each data point represents the average of two trials. The protein standard data fit the linear regression as follows: y = 12.335 − 4.26661x (R = 0.99494). Abs280, absorbance at 280 nm.

  • FIG. 5.
    FIG. 5.

    Overall structure of GCYH-IB. (A) Ribbon diagram showing a side view (left) and a top view (right) of the GCYH-IB biological tetramer. The two dimers are colored in shades of orange and blue, respectively. The four active centers harboring the metal binding sites and GTP binding pockets are located at the intersubunit interfaces and are indicated with arrowheads. Zinc ions, acetate ligands, and the conserved T-fold substrate anchor Glu216 in the active sites are shown as magenta balls, green Corey-Pauling-Koltun model, and red sticks, respectively. (B) Stereoview of a figure of merit (FOM)-weighted experimental electron density map (resolution, 2.2 Å; contour level, 1.5 σ), calculated after solvent flattening in the β-sheet region and superimposed on the refined model. The figure was prepared with Bobscript (15). (C) Ribbon diagram showing a side view (left) and a top view (right) of the homodecamer of GCYH-IA. The monomer is shown in red.

  • FIG. 6.
    FIG. 6.

    Topology of the GCYH-IB monomer and structural comparison with other T-fold enzymes. Topology diagrams of N. gonorrhoeae GCYH-IB (A), A. flavus urate oxidase (B), and E. coli GCYH-IA (C) calculated with the program TOPS (34). The N- and C-terminal modules of the core bimodular T-fold are shown in magenta and cyan, respectively. Cα trace superpositions of the N-terminal (magenta) and C-terminal (cyan) modules of GCYH-IB with corresponding enzymes (green) are shown in panels B and C. Secondary structure nomenclature is shown for the two isozymes.

  • FIG. 7.
    FIG. 7.

    Structure-guided multisequence alignment of GCYH-IA and GCYH-IB in the shared T-fold region. For clarity, only four sequences from each subfamily are shown. Residue labels and secondary structure elements (nomenclature same as in Fig. 6) are shown above and below the sequence alignment for subfamilies A and B, respectively. Residues conserved between the two subfamilies are in blue boxes, with the invariant residues (the substrate-binding Glu and metal-coordinating Cys) highlighted in red. Conserved regions within the GCYH-IB subfamily are in black boxes. The metal ion liganding side chains in each subfamily are labeled with red stars. The # symbol indicates a metal liganding side chain from the neighboring subunit. Additional GCYH-IB-specific, strictly conserved active-site residues are labeled with blue stars. Residues known to interact via their side chains or backbones with the GTP substrate in crystal structures of GCYH-IA are labeled with green stars and black asterisks, respectively. Ec, Escherichia coli; Tt, Thermus thermophilus; Hs, Homo sapien; Rn, Rattus norvegicus; Tm, Thermotoga maritima; Bs, Bacillus subtilis; Sa, Staphylococcus aureus; Ng, Neisseria gonorrhoeae.

  • FIG. 8.
    FIG. 8.

    The metal site in GCYH-IB. (A) Stereoview of annealed omit Fo-Fc electron density map (1,500 K, 2.2 Å, 2.5 σ; Zn2+ and its ligands omitted from the phase calculation) superimposed on the model. (B) Three-dimensional alignment of GCYH-IB (orange and cyan; labels in roman type) and T. thermophilus GCYH-IA (gray; italic and underlined labels) in the active-site region. Strictly conserved Cys149 in the metal binding loop of GCYH-IB is shown, although it does not interact with the bound metal. (C) Mn2+-occupied metal site in the GCYH-IB·Mn2+ complex. Stereoview of annealed omit Fo-Fc electron density map (1,500 K, 2.04 Å, 2.5 σ; Mn2+ and its ligands omitted from phase calculation) superposed on the model. Enzyme subunits are colored as in Fig. 5A. Metal ions and water molecules are shown as magenta and red spheres, respectively. Secondary structure elements in GCYH-IB are labeled. The figure was prepared in Bobscript (15).

  • FIG. 9.
    FIG. 9.

    Putative GTP binding pocket in GCYH-IB and comparison with GCYH-IA. (A) Docking model of the complex of N. gonorrhoeae GCYH-IB with 8-oxo-GTP shown in the active-site region. (B) Crystal structure of T. thermophilus GCYH-IA in complex with 8-oxo-GTP shown in the active-site region. Left panel shows the Corey-Pauling-Koltun model representation of the intersubunit interface showing the substrate binding cleft. Right panel shows stereoview of the substrate binding pocket showing interactions with the enzyme. Subunits are colored as in Fig. 5A. All panels depict the view of the intersubunit interface from the helical (outer) side of the barrel, i.e., looking down the arrows in Fig. 5A. Secondary structure elements mentioned in the text are labeled. 8-Oxo-GTP and the active-site side chains interacting with substrate are shown in stick model. The zinc ion is shown as a magenta ball.

Tables

  • TABLE 1.

    Distribution and candidate Zur-dependent regulation of alternative GCYH-I genes in bacteriaa

    OrganismcPresence of:
    folEfolE2
    Enterobacteria
        Escherichia coli +−
        Salmonella typhimurium +−
        Yersinia pestis +−
        Klebsiella pneumoniae b ++a
        Serratia marcescens ++a
        Erwinia carotovora +−
        Photorhabdus luminescens +−
        Proteus mirabilis +−
    Gammaproteobacteria
        Vibrio cholerae +−
        Acinetobacter sp. strain ADP1++a
        Pseudomonas aeruginosa ++a
        Pseudomonas entomophila L48++a
        Pseudomonas fluorescens Pf-5++a
        Pseudomonas syringae ++a
        Pseudomonas putida ++a
        Hahella chejuensis KCTC 2396++a
        Chromohalobacter salexigens DSM 3043++a
        Methylococcus capsulatus ++a
        Xanthomonas axonopodis ++a
        Xanthomonas campestris ++a
        Xylella fastidiosa ++a
         Idiomarina loihiensis −+
        Colwellia psychrerythraea ++
        Pseudoalteromonas atlantica T6c++a
        Pseudoalteromonas haloplanktis TAC125++
        Alteromonas macleodi +−
        Nitrosococcus oceani ++
        Legionella pneumophila +−
        Francisella tularensis +−
    Betaproteobacteria
        Chromobacterium violaceum +−
         Neisseria gonorrhoeae −+
        Burkholderia cepacia R18194++
        Burkholderia cenocepacia AU 1054++
        Burkholderia xenovorans +−
        Burkholderia mallei +−
         Bordetella pertussis −+
        Ralstonia eutropha JMP134+−
        Ralstonia metallidurans ++
        Ralstonia solanacearum +−
         Methylobacillus flagellatus −+
         Nitrosomonas europaea −+
        Azoarcus sp.++
    Bacilli/Clostridia
        Bacillus subtilis d ++
        Bacillus licheniformis ++
        Bacillus cereus +−
        Bacillus halodurans ++
        Bacillus clausii +−
        Geobacillus kaustophilus +−
         Oceanobacillus iheyensis −+
         Staphylococcus aureus −+

    • ↵ a Genes that are preceded by candidate Zur binding sites.

    • ↵ b Zur-regulated cluster is on the virulence plasmid pLVPK.

    • ↵ c Examples of organisms with no folE genes are in boldface type.

    • ↵ d Zn-dependent regulation of B. subtilis folE2 by Zur was experimentally verified (17).

  • TABLE 2.

    Metal dependence of B. subtilis GCYH-IB enzymatic activity

    MetalMetal concn (μM)aRelative activity (%)
    No metal00
    Mn(II)500100
    Fe(II)100075 ± 8
    Mg(II)10043 ± 4
    Co(II)10024 ± 3
    Zn(II)5014 ± 1
    Ni(II)1009.8 ± 1.3
    Ca(II)NA0
    Cd(II)NA0
    Cu(II)NA0
    Co(III)NA0
    Fe(III)NA0

    • ↵ a Metal concentration for optimal activity. NA, no activity detected regardless of metal concentration.

  • TABLE 3.

    X-ray data collection, phasing, and structure refinement statistics

    ParameterValue(s) for:
    GCYH-IBGCYH-IB·Mn2+
    Structure refinement
        Resolution range (Å)45.9-2.2h 30.33-2.04
        No. of reflections (working/free)42,833i/4,63131,937/1,710
        No. of atoms
            Protein/water3,829/1333,829/261
            Active-site metal ions22
            Other ions12
            Other ligands43
        R cryst/R free j 0.20/0.250.20/0.26
        rmsd bond length (Å)0.0070.006
        rmsd bond angle (°)0.8250.993
        Ramachandran plot—residues in:
            Favored regions (%)93.296.9
            Allowed regions (%)6.83.1
        Wilson B factor (Å2)40.337.4
    Data collection
        Space groupC2221 C2221
        Data setSepeak Seinflection Seremote
            Unit cell (Å)91.7, 100.3, 114.191.7, 100.2, 114.091.8, 100.4, 114.192.2, 100.4, 113.9
            Wavelength (Å)0.97930.97950.97470.9761
            Resolution (Å)50.0-2.2050.0-2.2050.0-2.2030.33-2.04
            Unique reflections26,718 (2,616)a 26,761 (2,656)26,490 (2,136)33,675 (3,314)
            Completeness (%)99.8 (99.0)99.8 (100.0)99.0 (92.0)99.8 (99.9)
            Redundancy5.7 (5.0)5.8 (5.7)5.4 (3.5)4.5 (4.5)
            R merge (%)b 8.7 (61.5)7.5 (36.9)8.4 (89.1)7.1 (42.8)
            I/σ(I)16.3 (2.4)17.6 (4.2)15.0 (3.5)11.0 (2.8)
    Phasing statistics (29-2.30 Å)
        R der c 0.074d 0.137
        R anom e 0.0690.0590.058
        R Cullis f (isod/ano)0.49/0.730.83/0.720.84/0.86
        Phasing powerg
            Centric0.060.110.16
            Acentric
                isod 0.060.090.15
                ano1.201.461.10
        FOM, overall0.34
            After DM0.83
            After Solomon0.85
        No. of Se sites15

    • ↵ a Highest-resolution shell (2.2-2.28 Å for GCYH-IB, and 2.04-2.11 Å for GCYH-IB·Mn2+) information in parentheses.

    • ↵ b R merge = 100 × (Σ h Σ i |I(h) − I(h) i |)/Σ h Σ iI(h) i , where I(h)i is the ith observation of reflection h and I(h) is the mean intensity of all observations of reflection h.

    • ↵ c R der = Σ h |F PH − F P|/Σ h |F P|, where |F P| and |F PH| are the observed structure factor amplitudes of the native and the derivative, respectively.

    • ↵ d The dispersive differences were treated as isomorphous replacement information where the data collected at a wavelength of 0.9747 Å are treated as native data.

    • ↵ e R anom = Σ h |F PH+ − F PH−|/Σ h |F PH|, where |F PH+| and |F PH−| are the Friedel pair observed structure factor amplitudes of the derivative at a given wavelength, and FPH is their average.

    • ↵ f R Cullis = Σ h [|F H| − (|F PH| − |F P|)]/Σ h (|F PH| − |F P|), where |F H| is the calculated heavy-atom structure factor for reflection h.

    • ↵ g PPdisp= (1/N refl)Σ h [||F PH| − |F P||/∫0 2 π(|F PH − F PH calc|) P(φ) d(φ)], where P(φ) is the probability of a phase value of φ for reflection h. PPano = (1/N refl)Σ h [|Δobs ANO|/∫0 2 π(|Δobs ANO − Δcalc ANO|) P(φ) d(φ)], where Δobs ANO and Δcalc ANO are the Friedel pair differences in the observed and calculated structure factor amplitudes, respectively, for reflection h.

    • ↵ h During density modification, structure factors were calculated for remote-wavelength data in the resolution range 2.3 to 2.2 Å.

    • ↵ i Anomalous pairs treated as separate reflections.

    • ↵ j Crystallographic R factor = 100 × (Σ h ||F obs(h)| − |F calc(h)||)/Σ h |F obs(h)|, where F obs(h) and F calc(h) are the observed structure factor amplitude and the structure factor amplitude calculated from the model, respectively. The free R factor was monitored with 10% and 5% of the data excluded from refinement for GCYH-IB and GCYH-IB·Mn2+, respectively.

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KEYWORDS

Bacterial Proteins
Folic Acid
GTP Cyclohydrolase
zinc

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