CP (capsular polysaccharide) is an important virulence factor during infections by the bacterium Staphylococcus aureus. The enzyme CapF is an attractive therapeutic candidate belonging to the biosynthetic route of CP of pathogenic strains of S. aureus. In the present study, we report two independent crystal structures of CapF in an open form of the apoenzyme. CapF is a homodimer displaying a characteristic dumb-bell-shaped architecture composed of two domains. The N-terminal domain (residues 1–252) adopts a Rossmann fold belonging to the short-chain dehydrogenase/reductase family of proteins. The C-terminal domain (residues 252–369) displays a standard cupin fold with a Zn2+ ion bound deep in the binding pocket of the β-barrel. Functional and thermodynamic analyses indicated that each domain catalyses separate enzymatic reactions. The cupin domain is necessary for the C3-epimerization of UDP-4-hexulose. Meanwhile, the N-terminal domain catalyses the NADPH-dependent reduction of the intermediate species generated by the cupin domain. Analysis by ITC (isothermal titration calorimetry) revealed a fascinating thermodynamic switch governing the attachment and release of the coenzyme NADPH during each catalytic cycle. These observations suggested that the binding of coenzyme to CapF facilitates a disorder-to-order transition in the catalytic loop of the reductase (N-terminal) domain. We anticipate that the present study will improve the general understanding of the synthesis of CP in S. aureus and will aid in the design of new therapeutic agents against this pathogenic bacterium.

INTRODUCTION

The proliferation of antibiotic resistance challenges our ability to effectively combat disease [1,2]. This situation is particularly problematic in connection with the opportunistic pathogen Staphylococcus aureus. S. aureus is a commensal bacterium residing in ~20% of the general population. Although generally harmless, S. aureus may cause life-threatening infections through sporadic breaches of the local defences [35]. In fact, S. aureus is a leading cause of nocosomial infections in hospitals worldwide [6]. Resistance to antibiotics of last resort such as methicillin and vancomycin further increases the risks associated with this bacterium [1,7,8]. It is thus desirable to boost the quality and quantity of our therapeutic arsenal against this pathogen.

CP (capsular polysaccharide) and its biosynthetic machinery are potentially useful therapeutic targets to combat S. aureus. CP is a polymeric carbohydrate attached to the outer surface of S. aureus that enhances pathogenesis by rendering the bacterium resistant to phagocytosis [914]. More than 70% of clinical isolates of S. aureus belong to either the CP5 or CP8 serotypes. These strains synthesize a polysaccharide containing repeating units of N-acetyl-L-fucosamine, N-acetyl-D-fucosamine and N-acetyl-D-mannosamine uronic acid.

The biosynthetic pathway of nucleotide-activated UDP-L-FucNAc (uridine diphosphate N-acetyl-L-fucosamine) is well conserved among several pathogenic bacteria producing CP, such as S. aureus, Pseudomonas aeruginosa, Staphylococcus pneumoniae and Bacteroides fragilis [15]. Mechanistic studies have shown that UDP-L-FucNAc is produced from the precursor UDP-D-GlcNAc (uridine diphosphate N-acetyl-D-glucosamine) through the sequential action of three enzymes CapE, CapF and CapG in S. aureus [15,16]. These three enzymes catalyse a total of five chemical transformations (Figure 1).

Synthesis of UDP-L-FucNAc in S. aureus

Figure 1
Synthesis of UDP-L-FucNAc in S. aureus

This pathway requires five chemical steps catalysed by three enzymes CapE, CapF and CapG. CapE catalyses 4,6-dehydrogenation of the substrate UDP-L-GlcNAc and C5-epimerization of intermediate 1. CapF catalyses C3-epimerization of intermediate 2 and NADPH-dependent reduction of intermediate 3 to yield product 4. Finally, CapG catalyses 2-epimerization of 4 resulting in the final product UDP-L-FucNAc. Full names of intermediate compounds are: intermediate 1, UDP-2-acetamido-2,6-dideoxy-α-D-xylo-4-hexulose; intermediate 2, UDP-2-acetamido-2,6-dideoxy-β-L-arabino-4-hexulose; intermediate 3, UDP-2-acetamido-2,6-dideoxy-β-L-lyxo-4-hexulose; and compound 4, UDP-2-acetamido-2,6-dideoxy-β-L-talose. The bonds marked with an asterisk indicate chemical groups subjected to enzymatic modification. This pathway was adapted from previous mechanistic studies using CapEFG and its close homologue system WbjBCD from P. aeruginosa [15,16].

Figure 1
Synthesis of UDP-L-FucNAc in S. aureus

This pathway requires five chemical steps catalysed by three enzymes CapE, CapF and CapG. CapE catalyses 4,6-dehydrogenation of the substrate UDP-L-GlcNAc and C5-epimerization of intermediate 1. CapF catalyses C3-epimerization of intermediate 2 and NADPH-dependent reduction of intermediate 3 to yield product 4. Finally, CapG catalyses 2-epimerization of 4 resulting in the final product UDP-L-FucNAc. Full names of intermediate compounds are: intermediate 1, UDP-2-acetamido-2,6-dideoxy-α-D-xylo-4-hexulose; intermediate 2, UDP-2-acetamido-2,6-dideoxy-β-L-arabino-4-hexulose; intermediate 3, UDP-2-acetamido-2,6-dideoxy-β-L-lyxo-4-hexulose; and compound 4, UDP-2-acetamido-2,6-dideoxy-β-L-talose. The bonds marked with an asterisk indicate chemical groups subjected to enzymatic modification. This pathway was adapted from previous mechanistic studies using CapEFG and its close homologue system WbjBCD from P. aeruginosa [15,16].

The first two enzymes CapE and CapF are proposed to belong to the SDR (short-chain dehydrogenase/reductase) superfamily of proteins. This idea is supported by sequence homology, and by prediction of a conserved catalytic triad and a classical nucleotide-binding motif GXXGXXG [15,17]. However, no high-resolution structural data for any of these enzymes has been reported so far. Detailed knowledge of their three-dimensional structures is needed to clarify their mechanism and function. Moreover, obtaining high-resolution structural information is a necessary step towards designing antibiotic therapeutics based on the inactivation of these enzymes.

In the present study, we report the new crystal structure of enzyme CapF obtained in two different space groups at resolutions of 2.45 Å (1 Å=0.1 nm) and 2.7 Å. The structure revealed that CapF is a homodimer arranged in a unique architecture not observed previously. CapF comprises a larger N-terminal region that adopts a typical SDR fold, and a smaller C-terminal region displaying a classical cupin motif with a bound Zn2+ metal ion. Mutational analysis demonstrated that each domain catalyses one enzymatic reaction: the cupin motif is responsible for an epimerization step, whereas the SDR domain catalyses the subsequent NADPH-dependent reduction.

EXPERIMENTAL

Expression and purification of untagged full-length CapF

Full-length SeMet (selenomethionine)-labelled and wild-type CapF were produced and purified as described previously [18]. Briefly, Escherichia coli Rosetta2 (DE3) cells expressing SeMet-labelled or wild-type protein were harvested, lysed by sonication and purified in a two-step procedure. First, we employed anion-exchange chromatography in a HiTrap Q XL column (GE Healthcare). Secondly, fractions containing CapF were subjected to gel-filtration chromatography in a HiLoad 26/60 Superdex 200 column (GE Healthcare) equilibrated with 50 mM Tris/HCl, pH 8.0, and 500 mM NaCl. Fractions containing CapF were concentrated with a 10 kDa Centriprep filtration unit (Millipore). Protein concentration was determined spectrophotometrically at 280 nm using the calculated molar extinction coefficient of CapF [19]. Protein was stored at −20°C in 40% (v/v) glycerol.

Expression and purification of CapF variants

Muteins and deletion constructs of CapF with a His6 tag at the N-terminal end were expressed in E. coli Rosetta2 (DE3) cells. A scheme with all of the constructs prepared in the present study is shown in Supplementary Figure S1 (at http://www.BiochemJ.org/bj/443/bj4430671add.htm). Cells were harvested by centrifugation at 7000 g for 10 min at 4°C, and washed with lysis buffer containing 50 mM Tris/HCl, pH 8.0, and 500 mM NaCl. Cells were disrupted on ice for 15 min using a sonicator (Tommy) set at 150 W. Cell lysate was centrifuged at 40000 g for 30 min at 4°C, and insoluble debris was discarded. Supernatant was loaded on to a HisTrap column (GE Healthcare) equilibrated with a buffer containing 50 mM Tris/HCl, pH 8.0, and 500 mM NaCl. Protein bound to the affinity column was initially washed with 20 ml of lysis buffer and was subsequently eluted with the same buffer supplemented with 0.5 M imidazole. Protein fractions containing CapF were further purified on a HiLoad 26/60 Superdex 200 gel-filtration chromatography column (GE Healthcare) equilibrated with a solution containing 50 mM Tris/HCl, pH 8.0, and 500 mM NaCl. Fractions of CapF were concentrated and stored as above. The UV–visible spectrum showed no evidence of NADPH bound to CapF.

Expression and purification of CapE

Full-length CapE with a His6 tag at the N-terminal end was expressed in E. coli Rosetta2 (DE3) cells, and purified as described for CapF above. CapE fractions were concentrated and stored at −20°C in a solution containing 40% (v/v) glycerol. Protein concentration was determined spectrophotometrically at 280 nm using the calculated molar extinction coefficient of the protein (ϵ=19200 M−1·cm−1).

Protein crystallization

Crystals of SeMet-labelled CapF suitable for X-ray diffraction analysis were obtained by the hanging drop method. We mixed 1 μl of fresh protein solution at 15 mg·ml−1 in 10 mM Tris/HCl, pH 8.0, and 1 μl of crystallization solution composed of 100 mM Mes, pH 7.2, 20% (v/v) glycerol, 100 mM (NH4)2SO4, 300 mM NaCl and 3.9 M sodium formate. Diamond-shaped crystals grew to an approximate size of 0.3×0.2×0.2 mm3 within 8 weeks. Alternatively, crystals of SeMet-labelled CapF were obtained in a solution containing 17.5% (w/v) PEG [poly(ethylene glycol)] 3350 and 0.2 M sodium malonate.

Data collection and phasing

Suitable crystals of CapF were identified, harvested and mounted under a stream of cold nitrogen (100 K) at beamline BL17A of the Photon Factory (Tsukuba, Japan). Three datasets each at a different wavelength were collected on the basis of the absorption edge of selenium: 0.9792 Å (peak, maximum f”); 0.9796 Å (edge, minimum f'); and 1.0000 Å (remote). Diffraction data were indexed and processed with HKL2000 software [20]. Phase determination and construction of an initial model was performed using the automated module of the SOLVE/RESOLVE program [2123].

Structure refinement

We refined data of SeMet-labelled crystals of CapF belonging to trigonal (P3221) and orthorhombic (C2221) space groups. Refinement of crystals of SeMet-labelled protein has been used as a surrogate for native protein in numerous structures (e.g. [2426]). Data were processed using MOSFLM [27], and were merged and scaled using the SCALA program of the CCP4 suite [28]. Atomic co-ordinates of CapF obtained using SOLVE/RESOLVE were used as an input model to determine the structure of CapF in the trigonal space group by molecular replacement with the program PHASER [29]. Data were further refined using REFMAC5 [30] with TLS (Translation/Libration/Screw) parameterization [31], and COOT [32]. During all of the refinement procedures SeMet replaced methionine residues. The refined structure of the trigonal crystal was used as the input model to determine the structure of CapF in the orthorhombic space group with PHASER [29]. Refinement was carried out as described for the trigonal crystal. Because the data of the orthorhombic crystal was collected at the peak of the anomalous scattering of selenium, coefficients f' and f” of selenium were incorporated in the refinement with REFMAC using separate Bijvoet pairs F+ and F [30]. Nonetheless, the contribution of anomalous scattering was minimal as demonstrated by the small statistical differences with respect to a mock refinement using averaged amplitudes [less than 0.6% for each Rfactor and Rfree, and RMSD (root mean square deviation) between equivalent atomic co-ordinates below 0.1 Å]. Model quality was assessed with PROCHECK [33]. The small difference between Rfree and Rfactor in the structure of the trigonal crystal (RfreeRfactor=2.2%) is probably explained by its very high solvent content (Supplementary Table S1 at http://www.BiochemJ.org/bj/443/bj4430671add.htm). Co-ordinate and structure factor files corresponding to refined models have been deposited in the PDB under accession codes 3ST7 and 3VHR.

Identification of Zn2+

The identity of the heavy metal ion bound in the cupin pocket was determined by X-ray anomalous diffraction analysis [3437]. This procedure required two datasets collected at a wavelength before (1.270 Å) and after (1.300 Å) the absorption edge of Zn2+. Each dataset was indexed using MOSFLM and scaled using SCALA to a resolution of 3.5 Å. We used PHASER to obtain an initial model of CapF, followed by refinement using REFMAC5 (until Rfree<24%). Anomalous difference electron density maps (Bijvoet-difference Fourier map) were calculated using programs SFALL, CAD, FFT and MAPMASK of the CCP4 suite [38].

Enzymatic assay

The CapF substrate was generated in situ by employing the enzyme CapE (which precedes CapF in the biosynthetic pathway of UDP-L-FucNAc). A typical enzymatic assay contained 1.7 mM substrate UDP-D-GlcNAc (Wako), 0.7 mM coenzyme NADPH or NADP+ (Wako), and enzymes CapE (His6-tagged) and CapF (untagged wild-type or His6-tagged muteins; Supplementary Figure S1) both at a concentration of 2 μM. The buffer used was 20 mM Tris/HCl, pH 8.0, and 10 mM MgCl2 in a total volume of 100 μl. Reaction mixtures were incubated at 37°C for 2 h. Enzymatic reactions were stopped by addition of 100 μl of ice-cold phenol/chloroform/isoamyl alcohol in a 25:24:1 molar ratio. Supernatant containing sugars were mixed with 100 μl of chloroform and analysed by HPLC using a CarboPac PA1 anion-exchange column (Dionex) as described previously [39]. Identification of monosaccharides was partly on the basis of previous reports [15,16]: substrate UDP-D-GlcNAc, 13.4 min; intermediate 2 UDP-2-acetamido-2,6-dideoxy-α-D-xylo-4-hexulose, 13.9 min; intermediate 3 UDP-2-acetamido-2,6-dideoxy-β-L-arabino-4-hexulose, 20.2 min; product UDP-2-acetamido-2,6-dideoxy-β-L-talose, 12.9 min; and coenzyme NADPH or NADP+, 17.9 min.

ITC (isothermal titration calorimetry)

Binding constants and thermodynamic parameters of the interaction between CapF and nucleotides NADPH or NAD+ were determined with an ITC200 instrument (GE Healthcare) at 25°C. Samples were equilibrated with a buffer composed of 20 mM Hepes and 150 mM NaCl at pH 8.0. Aliquots of a solution containing nucleotide NADPH at a concentration of 500 μM were injected stepwise into a cell filled with a solution of CapF at a concentration of 50 μM. The concentrations of NADP+ and protein in the titration with NADP+ were increased to 3.80 mM and 96 μM respectively to observe the binding isotherm under low-affinity conditions [40]. Heat generated from the dilution of NADPH and protein was negligible, but that of NADP+ had to be subtracted prior to data analysis. Titration curves were fitted to a one-site binding isotherm [41].

DSC (differential scanning calorimetry)

The thermal stability of CapF was determined in a VP-capillary calorimeter (GE Healthcare). Protein samples at a concentration of 50 μM were equilibrated in a solution containing 50 mM Hepes, pH 8.0, and 200 mM NaCl. Thermograms were recorded between 283 K and 373 K at a rate of 1 K·min−1. The buffer baseline was subtracted and data were normalized by protein concentration using Origin software.

RESULTS AND DISCUSSION

CapF adopts a unique two-domain architecture

The crystal structure of CapF in trigonal space group P3221 was solved by the multi-wavelength anomalous diffraction method and refined to a resolution of 2.45 Å (Table 1). The asymmetric unit consisted of a single polypeptide chain (residues 1–369). CapF adopted a characteristic dumb-bell-shaped architecture comprising two domains. The N-terminal domain included residues Met1 to Pro251, whereas the C-terminal domain was composed of residues Leu252 to Val369 (Figure 2A).

Table 1
Data collection, phasing, and refinement statistics

Statistical values in parentheses refer to the highest-resolution bin. FOM, figure of merit.

 Remote* Peak* Edge* Refined 1 Refined 2 
Data collection      
 Space group P3221 P3221 P3221 P3221 C2221 
 Unit cell      
 Dimensions (Å) a=b=119.73, c=129.43 a=b=119.58, c=129.21 a=b=119.57, c=129.25 a=b=119.53, c=129.45 a=66.06, b=105.01, c=131.53 
 Angles (°) α=β=90, γ=120 α=β=γ=90    
 Wavelength (Å) 1.000 0.97922 0.97962 1.000 0.9793 
 Resolution range (Å) 50–2.80 50–2.90 50–2.80 40–2.45 50–2.70 
 Total observations 281357 248846 280945 351690 88858 
 Unique observations 26838 24154 26795 39695 12933 
 I/σI 15.4 (4.8) 12.4 (4.4) 14.4 (4.7) 20 (2.8) 13.4 (4.0) 
 Completeness (%) 100 (100) 100 (100) 100 (100) 99.8 (100) 100 (100) 
Rmerge (%) 6.2 (34.4) 8.6 (40.0) 6.5 (34.6) 7.0 (86.0) 9.9 (46.7) 
 Multiplicity 10.5 (9.9) 10.3 (9.6) 10.5 (9.9) 8.9 (9.0) 6.9 (7.0) 
Phasing      
 Sites     
 FOM (solve)  0.32    
 FOM (resolve)  0.65    
Refinement      
Rwork/Rfree (%)    19.2/21.4 19.8/25.9 
 Solvent content (%)    81 55 
 Number of protein chains    
 Number of protein residues    369 342 
 Number of zinc atoms    
 Number of water molecules    60 28 
 B-factor, protein (Å2   70.6 47.3 
 B-factor, zinc (Å2   64.9 35.0 
 B-factor, water (Å2   61.2 33.0 
 Solvent content (%)    81 55 
 RMSD bonds (Å)    0.016 0.008 
 RMSD bonds (°)    1.70 1.30 
 Co-ordinate error (Å)    0.16 0.34 
Ramachandran plot      
 Preferred regions (%)    90.5 90.4 
 Allowed regions (%)    9.5 9.6 
 Outliers (%)    
 PDB code    3ST7 3VHR 
 Remote* Peak* Edge* Refined 1 Refined 2 
Data collection      
 Space group P3221 P3221 P3221 P3221 C2221 
 Unit cell      
 Dimensions (Å) a=b=119.73, c=129.43 a=b=119.58, c=129.21 a=b=119.57, c=129.25 a=b=119.53, c=129.45 a=66.06, b=105.01, c=131.53 
 Angles (°) α=β=90, γ=120 α=β=γ=90    
 Wavelength (Å) 1.000 0.97922 0.97962 1.000 0.9793 
 Resolution range (Å) 50–2.80 50–2.90 50–2.80 40–2.45 50–2.70 
 Total observations 281357 248846 280945 351690 88858 
 Unique observations 26838 24154 26795 39695 12933 
 I/σI 15.4 (4.8) 12.4 (4.4) 14.4 (4.7) 20 (2.8) 13.4 (4.0) 
 Completeness (%) 100 (100) 100 (100) 100 (100) 99.8 (100) 100 (100) 
Rmerge (%) 6.2 (34.4) 8.6 (40.0) 6.5 (34.6) 7.0 (86.0) 9.9 (46.7) 
 Multiplicity 10.5 (9.9) 10.3 (9.6) 10.5 (9.9) 8.9 (9.0) 6.9 (7.0) 
Phasing      
 Sites     
 FOM (solve)  0.32    
 FOM (resolve)  0.65    
Refinement      
Rwork/Rfree (%)    19.2/21.4 19.8/25.9 
 Solvent content (%)    81 55 
 Number of protein chains    
 Number of protein residues    369 342 
 Number of zinc atoms    
 Number of water molecules    60 28 
 B-factor, protein (Å2   70.6 47.3 
 B-factor, zinc (Å2   64.9 35.0 
 B-factor, water (Å2   61.2 33.0 
 Solvent content (%)    81 55 
 RMSD bonds (Å)    0.016 0.008 
 RMSD bonds (°)    1.70 1.30 
 Co-ordinate error (Å)    0.16 0.34 
Ramachandran plot      
 Preferred regions (%)    90.5 90.4 
 Allowed regions (%)    9.5 9.6 
 Outliers (%)    
 PDB code    3ST7 3VHR 
*

Data were collected on the same crystal at three different wavelengths.

Crystal structure of CapF

Figure 2
Crystal structure of CapF

(A) Stereo view of the monomer of CapF as observed in the asymmetric unit. Secondary elements belonging to the N-terminal (reductase or SDR) domain are depicted in blue; the C-terminal (cupin) domain is depicted in red. Active-site residues of the SDR domain (Ser94, Tyr103 and Lys107) are shown in yellow. The Zn2+ atom bound to the cupin domain (green sphere) and residues His290, Glu295 and His337 belonging to its co-ordination sphere are also shown. (B) Semi-transparent surface representation of the CapF homodimer. The colour scheme of monomer 1 is the same as above. The second molecule of CapF is depicted in light grey. White arrows indicate the positions of the reductase and the Zn2+-binding sites in monomer 1.

Figure 2
Crystal structure of CapF

(A) Stereo view of the monomer of CapF as observed in the asymmetric unit. Secondary elements belonging to the N-terminal (reductase or SDR) domain are depicted in blue; the C-terminal (cupin) domain is depicted in red. Active-site residues of the SDR domain (Ser94, Tyr103 and Lys107) are shown in yellow. The Zn2+ atom bound to the cupin domain (green sphere) and residues His290, Glu295 and His337 belonging to its co-ordination sphere are also shown. (B) Semi-transparent surface representation of the CapF homodimer. The colour scheme of monomer 1 is the same as above. The second molecule of CapF is depicted in light grey. White arrows indicate the positions of the reductase and the Zn2+-binding sites in monomer 1.

At three-dimensional search in the DALI server [42] found high homology between the N-terminal domain of CapF and the SDR superfamily of proteins (Supplementary Table S2 at http://www.BiochemJ.org/bj/443/bj4430671add.htm) [43,44]. SDR enzymes are characterized by a nucleotide-binding Rossmann motif [45,46]. An analogous analysis showed that C-terminal domain adopted a cupin fold [47,48]. The cupin domain of CapF displayed a heavy metal atom bound at the bottom of the β-barrel. We unambiguously identified the metal as Zn2+ by X-ray anomalous scattering analysis (Supplementary Figure S2 at http://www.BiochemJ.org/bj/443/bj4430671add.htm). Although Rossmann and cupin domains are ubiquitous in nature with more than 45000 members of each family in the Pfam database [49], the structure of CapF described in the present study constitutes, to the best of our knowledge, the first documented example of a protein combining both motifs within the same polypeptide chain.

Crystallographic symmetry analysis revealed that CapF forms a homodimer stabilized by a very large interaction surface (Figure 2B). Analysis of the contact interface in the PISA server [50] indicated that CapF buries 6800 Å2 of surface area upon dimer formation (3400 Å2 from each monomer). This extensive surface is composed mostly of residues of the C-terminal domain (2124 Å2, 63% of the total). The N-terminal domain contributed with a smaller interaction surface (1276 Å2, 37%). Protein dimerization was verified by gel-filtration chromatography, confirming that CapF is also a homodimer in solution (Supplementary Figure S3 at http://www.BiochemJ.org/bj/443/bj4430671add.htm).

Active site of the reductase domain is disordered in a second crystal structure

We determined a second crystal structure of CapF in the orthorhombic space group C2221 at a resolution of 2.7 Å (Table 1). This independent structure superimposed well on the trigonal structure, achieving an average RMSD value of 0.84 Å over 1371 mainchain atoms (Figure 3). The contribution of each domain to the dimer interface was nearly identical with that in the trigonal crystal (interaction surface=3336 Å2; cupin, 63%; SDR, 37%). A loop of the SDR domain containing residues Gly55 to Phe65 could not be traced in the electron density maps of the orthorhombic crystal. The probable reason was that these residues clashed with a neighbouring protein chain of an adjacent unit cell of the crystal. These same residues displayed the highest temperature factors in the trigonal structure, indicating a propensity to become disordered (Figure 4). Other SDR proteins such as RmlB [51] or the dehydrogenase domain of ArnA [52] undergo order–disorder transition of selected loops (not belonging to the catalytic site) in response to cofactor and/or substrate binding.

Structural differences between two independent crystals of CapF are localized at specific surface loops

Figure 3
Structural differences between two independent crystals of CapF are localized at specific surface loops

Comparison between the structure of the monomer of CapF crystallized in the trigonal P3221 space group (depicted in blue and red) and that crystallized in the orthorhombic C2221 space group (depicted in grey). Atomic co-ordinates were superimposed using the least squares method implemented in the program COOT [32]. Purple sticks represent residues belonging to loop Gly54→Ser66 and active-site loop Ser94→Gly110, both missing in the electron density of the C2221 crystal structure. Orange arrows point to the regions missing in the orthorhombic structure. The residues depicted in the Figure belong to the trigonal structure.

Figure 3
Structural differences between two independent crystals of CapF are localized at specific surface loops

Comparison between the structure of the monomer of CapF crystallized in the trigonal P3221 space group (depicted in blue and red) and that crystallized in the orthorhombic C2221 space group (depicted in grey). Atomic co-ordinates were superimposed using the least squares method implemented in the program COOT [32]. Purple sticks represent residues belonging to loop Gly54→Ser66 and active-site loop Ser94→Gly110, both missing in the electron density of the C2221 crystal structure. Orange arrows point to the regions missing in the orthorhombic structure. The residues depicted in the Figure belong to the trigonal structure.

Temperature factors (sausage representation) of CapF crystallized in the trigonal P3221 space group

Figure 4
Temperature factors (sausage representation) of CapF crystallized in the trigonal P3221 space group

Thickness is proportional to B-factor values. Red colour indicates ‘hotter’ (higher) B-factor values. Blue colour indicates residues with ‘colder’ (lower) B-factor values. Arrows indicate the position of disordered loops in the orthorhombic crystal.

Figure 4
Temperature factors (sausage representation) of CapF crystallized in the trigonal P3221 space group

Thickness is proportional to B-factor values. Red colour indicates ‘hotter’ (higher) B-factor values. Blue colour indicates residues with ‘colder’ (lower) B-factor values. Arrows indicate the position of disordered loops in the orthorhombic crystal.

Strikingly, the loop containing the predicted catalytic residues Tyr103 and Lys107 of the reductase domain was also disordered in the orthorhombic structure (Supplementary Figure S4 at http://www.BiochemJ.org/bj/443/bj4430671add.htm). Specifically, the residues between Ser95 and Gln109 could not be traced in the difference electron density (FoFc) maps. We did not find evidence of clashes between this loop and other regions of CapF in the crystal structure that could explain the dynamic disorder. The high mobility of this loop clearly contrasted with the orderly electron density features seen in the same loop of CapF crystallized in the trigonal space group (Supplementary Figure S4).

This interesting observation suggested that the active site of CapF undergoes a large structural remodelling during its catalytic cycle. To the best of our knowledge a similar order–disorder transition in the active site of this family of proteins has not been described previously. These fluctuations could be related to the unique primary sequence of CapF in this region compared with similar SDR proteins. In fact, the catalytic loop of CapF comprises between 16 and 31 fewer residues than that of homologous enzymes (Supplementary Figure S4). The relative smaller size of this region of CapF could facilitate an enhanced dynamic behaviour in response to external stimuli such as substrate and/or coenzyme binding.

Cofactor is needed only in the second reaction catalysed by CapF

Armed with the valuable structural information above, we embarked on a detailed structure–function analysis of CapF. Enzymatic assays were first employed to assess the cofactor requirements of CapF. We note that the enzyme CapE was employed to generate the substrate of CapF in situ (see Figure 1). HPLC chromatograms are shown in Figure 5, and integrated data are displayed in Table 2.

CapF requires coenzyme NADPH only for the reduction reaction

Figure 5
CapF requires coenzyme NADPH only for the reduction reaction

Each panel shows representative HPLC traces after incubation of the substrate UDP-D-GlcNAc with (A) NADPH, (B) CapE and NADPH, (C) CapE and CapF, (D) CapE, CapF and NADP+ and (E) CapE, CapF and NADPH. Arrows indicate the positions of the main peaks (P, product; S, substrate; Int2, intermediate 2; Int3, intermediate 3). The major peaks after reaction are shaded according to their identity: product (compound 4 in Figure 1) UDP-2-acetamido-2,6-dideoxy-β-L-talose, black; substrate, UDP-D-GlcNAc, light grey; intermediate 2, UDP-2-acetamido-2,6-dideoxy-β-L-arabino-4-hexulose, dark grey; intermediate 3, UDP-2-acetamido-2,6-dideoxy-β-L-lyxo-4-hexulose, mid grey. Chromatograms were normalized for the preparation of this Figure. The highest peak was given an arbitrary value of 100. To facilitate their comparison, the values of the combined peak sizes of substrate, intermediate 2, intermediate 3 and product are shown for each chromatogram (prior to normalization, in detector units, d.u.). Complete conversion into product was not observed because NADPH was the limiting reactant.

Figure 5
CapF requires coenzyme NADPH only for the reduction reaction

Each panel shows representative HPLC traces after incubation of the substrate UDP-D-GlcNAc with (A) NADPH, (B) CapE and NADPH, (C) CapE and CapF, (D) CapE, CapF and NADP+ and (E) CapE, CapF and NADPH. Arrows indicate the positions of the main peaks (P, product; S, substrate; Int2, intermediate 2; Int3, intermediate 3). The major peaks after reaction are shaded according to their identity: product (compound 4 in Figure 1) UDP-2-acetamido-2,6-dideoxy-β-L-talose, black; substrate, UDP-D-GlcNAc, light grey; intermediate 2, UDP-2-acetamido-2,6-dideoxy-β-L-arabino-4-hexulose, dark grey; intermediate 3, UDP-2-acetamido-2,6-dideoxy-β-L-lyxo-4-hexulose, mid grey. Chromatograms were normalized for the preparation of this Figure. The highest peak was given an arbitrary value of 100. To facilitate their comparison, the values of the combined peak sizes of substrate, intermediate 2, intermediate 3 and product are shown for each chromatogram (prior to normalization, in detector units, d.u.). Complete conversion into product was not observed because NADPH was the limiting reactant.

Table 2
Coenzyme NADPH is essential only for the reduction reaction

Results show the average±S.D. for three or more determinations. N.D., not detected.

Sample Substrate (%) Intermediate 2 (%) Intermediate 3 (%) Product (%) 
NADPH 100 N.D. N.D. N.D. 
CapE+NADPH 2.4±2.3 71.1±5.0 26.5±2.7 N.D. 
CapE+CapF 1.1±0.5 15.5±0.2 83.5±9.4 N.D. 
CapE+CapF+NADP+ 1.0±0.4 17.0±1.0 82.0±1.9 N.D. 
CapE+CapF+NADPH N.D. 12.5±0.9 42.8±3.7 44.6±3.7 
Sample Substrate (%) Intermediate 2 (%) Intermediate 3 (%) Product (%) 
NADPH 100 N.D. N.D. N.D. 
CapE+NADPH 2.4±2.3 71.1±5.0 26.5±2.7 N.D. 
CapE+CapF 1.1±0.5 15.5±0.2 83.5±9.4 N.D. 
CapE+CapF+NADP+ 1.0±0.4 17.0±1.0 82.0±1.9 N.D. 
CapE+CapF+NADPH N.D. 12.5±0.9 42.8±3.7 44.6±3.7 

Control experiments showed that the substrate UDP-D-GlcNAc was not modified when incubated with NADPH alone (Figure 5A). However, catalytic amounts of enzyme CapE with or without coenzymes generated a new major peak corresponding to intermediate 2 (only the reaction with NADPH is shown; Figure 5B). We also observed a minor peak at higher retention times that was tentatively assigned to intermediate 3. Appearance of this minor peak was probably caused by enolization of intermediate 2, similar to what occurs with the homologous protein FlaA1 [53]. However, we cannot completely exclude that this peak corresponded to a reaction by-product.

The presence of enzymes CapE and CapF in the reaction mixture (with or without oxidized coenzyme NADP+) resulted in a major peak at high retention times, which we interpreted as epimerization of intermediate 2 at the C3 position (Figures 5C and 5D). Finally, when the substrate was incubated with the three active components CapE, CapF and NADPH, it generated the expected product UDP-2-acetamido-2,6-dideoxy-β-L-talose (Figure 5E). The yield was quantitative within experimental error (mean±S.D.=44.6±3.7%) when taking into consideration that NADPH was the limiting reactant in our enzymatic assay (the molar concentration of NADPH was 41% that of the substrate UDP-D-GlcNAc). The appearance of product was coupled to consumption of intermediate 3, but not of intermediate 2.

Binding of NADPH is enthalpy-driven

We have shown above that the reduction step required one equivalent of NADPH. However, because neither NADPH nor NADP+ were observed in the crystal structure of CapF, we determined their binding properties by ITC (Figure 6 and Table 3).

Binding of coenzyme is modulated by its redox state

Figure 6
Binding of coenzyme is modulated by its redox state

CapF was titrated with (A) NADPH or (B) NADP+, at a temperature of 25°C. Upper panels correspond to titration kinetics. Lower panels show the integrated binding isotherms. Molar ratio refers to protein monomer. The binding isotherm of CapF titrated with NADP+ was corrected with the heat of dilution of the oxidized coenzyme before analysis. Correction was not necessary in (A) because the heat of dilution was negligible. The binding enthalpy (ΔH) and dissociation constant (Kd) were obtained by non-linear regression of the integrated data to a one-site binding model.

Figure 6
Binding of coenzyme is modulated by its redox state

CapF was titrated with (A) NADPH or (B) NADP+, at a temperature of 25°C. Upper panels correspond to titration kinetics. Lower panels show the integrated binding isotherms. Molar ratio refers to protein monomer. The binding isotherm of CapF titrated with NADP+ was corrected with the heat of dilution of the oxidized coenzyme before analysis. Correction was not necessary in (A) because the heat of dilution was negligible. The binding enthalpy (ΔH) and dissociation constant (Kd) were obtained by non-linear regression of the integrated data to a one-site binding model.

Table 3
Thermodynamic analysis of binding of coenzyme to CapF

Results show the average±S.D.

Coenzyme Kd (μM) ΔG (kcal·mol−1ΔH (kcal·mol−1TΔS (kcal·mol−1)* n 
NADPH 1.6±0.2 −7.9±0.2 −16.7±0.2 8.8 0.91±0.01 
NADP+ 61±6.1 −5.8±0.8 −9.8±0.8 4.0 0.86±0.06 
Coenzyme Kd (μM) ΔG (kcal·mol−1ΔH (kcal·mol−1TΔS (kcal·mol−1)* n 
NADPH 1.6±0.2 −7.9±0.2 −16.7±0.2 8.8 0.91±0.01 
NADP+ 61±6.1 −5.8±0.8 −9.8±0.8 4.0 0.86±0.06 
*

The temperature was 298 K.

Titration of CapF with the reduced coenzyme NADPH gave rise to a typical sigmoid binding isotherm. Thermodynamic parameters indicated that binding of NADPH to CapF was driven by a very favourable enthalpy change (ΔH=−16.7±0.2 kcal·mol−1; 1 kcal=4.184 kJ) and displayed moderate affinity (Kd=1.6±0.2 μM). Binding of NADPH to CapF was strongly opposed by the entropy term (−TΔS=8.8 kcal·mol−1), which accounted for approximately half of the absolute value of the enthalpy change. One monomer of CapF binds one molecule of NADPH (n=0.91±0.01).

The binding isotherm of NADP+ was characterized by markedly lower affinity for the apoenzyme. The dissociation constant determined for NADP+ (Kd=61±6.1 μM) was ~40-fold higher (lower affinity) than that for NADPH. The enthalpy change was significantly reduced in absolute terms (ΔH=−9.8±0.8 kcal·mol−1). Binding of NADP+ to CapF was also opposed by an unfavourable entropy term (−TΔS=4.0 kcal·mol−1). Overall, the affinities of both NADPH and NADP+ were smaller than those reported for similar enzymes [54,55], which explains why NADPH or NADP+ were not found in the crystal structures of CapF.

Importantly, the affinity gap between NADPH and NADP+ resulted in −2.1 kcal·mol−1 of free energy that can be utilized by the enzyme in each catalytic cycle. We propose that the order–disorder transition observed in the active-site loop of the SDR domain (Figure 4 and Supplementary Figure S4) could be coupled to the thermodynamic switch described just above. We expect that new crystal structures of CapF with either NADPH or NADP+ will reveal the identity of the residues interacting with the coenzyme.

Identification of catalytic domains

Two different constructs of His6–CapF, the first one comprising residues of the reductase domain (residues 1–243; CapFREDUCTASE) and the second one composed of residues of the cupin domain (residues 252–369; CapFCUPIN), were cloned, expressed and purified to homogeneity. Each construct was assayed in the presence of substrate, CapE and NADPH (Table 4). However, none of these two constructs were active when acting separately, or when added together into the reaction mixture. The structural integrity of each domain, evaluated by CD, ruled out that enzymatic inactivation was caused by large-scale unfolding (Supplementary Figure S5 at http://www.BiochemJ.org/bj/443/bj4430671add.htm) [56]. These experiments demonstrated that the intact topology of the native dimer was needed to preserve catalytic prowess in CapF.

Table 4
Mutational analysis of CapF

CapE, NADPH and UDP-D-GlcNAc were present in all assays. Results show the average±S.D. for three or more determinations. N.D., not detected.

Construct(s) used Substrate (%) Intermediate 2 (%) Intermediate 3 (%) Product (%) 
No CapF 2.4±2.3 71.1±5.0 26.5±2.7 N.D. 
Wild-type N.D. 12.5±0.9 42.8±3.7 44.6±3.7 
CapFREDUCTASE 2.3±0.1 71.5±7.6 25.7±1.8 2.3±0.1 
CapFCUPIN 1.1±0.4 72.2±8.4 26.7±1.5 N.D. 
CapFREDUCTASE and CapFCUPIN 1.8±0.4 69.3±8.2 28.3±4.1 0.6±0.1 
H290L 6.1±0.7 55.5±4.9 35.2±3.9 3.2±0.3 
F297Y N.D. 10.0±1.1 44.8±1.8 45.2±4.1 
T364Y N.D. 20.6±0.7 33.4±3.3 45.9±1.0 
S94A/Y103A 1.1±0.4 17.5±1.4 81.4±4.8 N.D. 
S94A/Y103A and CapFREDUCTASE N.D. 11.5±1.5 45.0±3.8 43.5±3.5 
Construct(s) used Substrate (%) Intermediate 2 (%) Intermediate 3 (%) Product (%) 
No CapF 2.4±2.3 71.1±5.0 26.5±2.7 N.D. 
Wild-type N.D. 12.5±0.9 42.8±3.7 44.6±3.7 
CapFREDUCTASE 2.3±0.1 71.5±7.6 25.7±1.8 2.3±0.1 
CapFCUPIN 1.1±0.4 72.2±8.4 26.7±1.5 N.D. 
CapFREDUCTASE and CapFCUPIN 1.8±0.4 69.3±8.2 28.3±4.1 0.6±0.1 
H290L 6.1±0.7 55.5±4.9 35.2±3.9 3.2±0.3 
F297Y N.D. 10.0±1.1 44.8±1.8 45.2±4.1 
T364Y N.D. 20.6±0.7 33.4±3.3 45.9±1.0 
S94A/Y103A 1.1±0.4 17.5±1.4 81.4±4.8 N.D. 
S94A/Y103A and CapFREDUCTASE N.D. 11.5±1.5 45.0±3.8 43.5±3.5 

To further investigate the enzymatic mechanism of CapF, we prepared two different muteins of full-length CapF containing point mutations in key residues: (i) at the catalytic site of the SDR domain (mutein S94A/Y103A); and (ii) belonging to the co-ordination sphere of Zn2+ (H290L). These constructs maintained a native-like dimeric conformation in solution, as indicated by size-exclusion chromatography (results not shown). Their enzymatic activity was measured as described above (Table 4 and Figure 7).

Mutational analysis

Figure 7
Mutational analysis

Representative HPLC traces after reaction (A) without CapF, (B) with wild-type CapF, (C) with variant H290L, (D) with variant S94A/Y103A and (E) with variant S94A/Y103A and CapFREDUCTASE. Substrate UDP-D-GlcNAc, CapE, and coenzyme NADPH were present in every reaction (AE). Normalization and integration values were obtained as in Figure 5. d.u., detector units; Int2, intermediate 2; Int3, intermediate 3; P, product.

Figure 7
Mutational analysis

Representative HPLC traces after reaction (A) without CapF, (B) with wild-type CapF, (C) with variant H290L, (D) with variant S94A/Y103A and (E) with variant S94A/Y103A and CapFREDUCTASE. Substrate UDP-D-GlcNAc, CapE, and coenzyme NADPH were present in every reaction (AE). Normalization and integration values were obtained as in Figure 5. d.u., detector units; Int2, intermediate 2; Int3, intermediate 3; P, product.

We noticed that none of these two muteins generated a final product. However, as shown below, their mechanism differed greatly. The HPLC elution profile of mutein H290L resembled that of assays without CapF, indicating that this mutein was completely inactive (compare Figures 7A and 7C). However, the activity of mutein S94A/Y103A resembled that of native CapF in the absence of coenzyme NADPH, i.e. it catalysed the reaction to intermediate 3, but not the reduction step because the SDR domain was catalytically dead (compare Figures 7D, 5C and 5D).

Importantly, CapFREDUCTASE rescued the full enzymatic activity of mutein S94A/Y103A (Figure 7E). When these two constructs are together in the reaction mixture, the intermediate 3 species generated by mutein S94A/Y103A (at its intact cupin domain) was properly processed by variant CapFREDUCTASE. We concluded that the catalytic potential of SDR remained intact even when the cupin domain was absent from the primary sequence. In contrast, the lack of activity of the CapFCUPIN construct suggested that this catalytic unit is very sensitive to changes in the architecture of the overall enzyme.

The integrity of the Zn2+-binding pocket of the cupin domain influenced the overall thermostability of the enzyme (Figures 8A and 8B). Removal of Zn2+ after incubation with 1.0 mM EDTA diminished the overall stability of the protein by 15°C (Figure 8C). The stability of CapF was recovered upon reconstitution with Zn2+. Similarly, mutein H337L and mutein H288A/H209A (both constructs modified the direct co-ordination sphere of Zn2+) suffered from diminished thermal stability (Supplementary Figure S6 at http://www.BiochemJ.org/bj/443/bj4430671add.htm). We also note that muteins F297Y and T364Y of the second co-ordination sphere of Zn2+ (Figure 8A) exerted a minor effect on the activity of CapF (Table 4), in contrast with the full inactivation observed in H290L.

Analysis of Zn2+-binding site

Figure 8
Analysis of Zn2+-binding site

(A) Overall view of the Zn2+-binding site in the cupin domain. Zn2+ is shown as a green sphere. Residues belonging to the Zn2+ co-ordination sphere His288, His290, Glu295 and His337, and also Phe297 and Thr364 (each replaced with a tyrosine residue in the mutational analysis), are shown. Broken lines represent co-ordination bonds between protein and Zn2+. A σA-weighted 2FoFc electron density map contoured at 12.0 σ is shown (magenta) to emphasize the location of Zn2+. Secondary elements are depicted in semi-transparent red. (B) Close-up view of the Zn2+-binding site. Co-ordination bonds with distances between each residue and Zn2+ are indicated. The electron density is same as in (A). (C) Integrity of the Zn2+-binding site is essential for thermal stability. Untreated CapF (black), CapF treated with 1 mM EDTA (red) or CapF pre-treated with 1 mM EDTA followed by reconstitution with Zn2+ (blue) were heated from 283 K to 373 K at a rate of 1 K·min−1. Heat capacity (Cp) was corrected with the buffer baseline and normalized by protein concentration (50 μM).

Figure 8
Analysis of Zn2+-binding site

(A) Overall view of the Zn2+-binding site in the cupin domain. Zn2+ is shown as a green sphere. Residues belonging to the Zn2+ co-ordination sphere His288, His290, Glu295 and His337, and also Phe297 and Thr364 (each replaced with a tyrosine residue in the mutational analysis), are shown. Broken lines represent co-ordination bonds between protein and Zn2+. A σA-weighted 2FoFc electron density map contoured at 12.0 σ is shown (magenta) to emphasize the location of Zn2+. Secondary elements are depicted in semi-transparent red. (B) Close-up view of the Zn2+-binding site. Co-ordination bonds with distances between each residue and Zn2+ are indicated. The electron density is same as in (A). (C) Integrity of the Zn2+-binding site is essential for thermal stability. Untreated CapF (black), CapF treated with 1 mM EDTA (red) or CapF pre-treated with 1 mM EDTA followed by reconstitution with Zn2+ (blue) were heated from 283 K to 373 K at a rate of 1 K·min−1. Heat capacity (Cp) was corrected with the buffer baseline and normalized by protein concentration (50 μM).

Comparison with RmlC and RmlD

The present study has revealed clear parallelisms between CapF and the pair of enzymes RmlC/RmlD. These enzymes catalyse similar sequential reactions to those of CapF, but in the biosynthetic pathway of L-rhamnose (a diastereoisomer of L-fucose). Specifically, RmlC catalyses a double C5,C3-epimerization reaction [5761], whereas RmlD is responsible for the subsequent NADPH-dependent reduction of the dTDP-4-hexulose species generated by RmlC [62,63].

Although the overall three-dimensional structure of RmlC and the cupin domain of CapF are very similar (RMSD=1.7 Å over 95 residues) and the substrate of RmlC fits nicely in the pocket of CapF, their constellations of catalytic residues differed substantially from each other (Figure 9A and Supplementary Figure S7 at http://www.BiochemJ.org/bj/443/bj4430671add.htm). RmlC does not display a Zn2+ ion (or metals) in the pocket of the β-barrel motif. Likewise, Zn2+ (or metals) are not required for enzymatic activity. All of these differences precluded unambiguous identification of the catalytic residues of the cupin domain based exclusively on structural comparisons.

Comparison between CapF and the homologue enzymes RmlC and RmlD

Figure 9
Comparison between CapF and the homologue enzymes RmlC and RmlD

(A) Superposition of the cupin domain of CapF with RmlC. (B) Superposition of the reductase domain of CapF with RmlD. Secondary structure of CapF is depicted in dark grey, and that of RmlC and RmlD in light grey. The molecule depicted in cyan corresponds to the substrate analogue dTNP-L-rhamnose bound to the active site of RmlC or RmlD. The Zn2+ ion and residues of its co-ordination sphere are shown in green and orange respectively. Black arrows indicate structural motifs not present in the cupin domain of CapF. Tyr128 (blue) and NADPH (yellow) of RmlD, and Tyr103 (orange) of the SDR domain of CapF are depicted in (B). The curved arrow suggests a mechanism of closure of Loop55–72 of CapF. The co-ordinates of RmlC and RmlD were retrieved from the PDB under accession codes 2IXH and 1KC3 respectively.

Figure 9
Comparison between CapF and the homologue enzymes RmlC and RmlD

(A) Superposition of the cupin domain of CapF with RmlC. (B) Superposition of the reductase domain of CapF with RmlD. Secondary structure of CapF is depicted in dark grey, and that of RmlC and RmlD in light grey. The molecule depicted in cyan corresponds to the substrate analogue dTNP-L-rhamnose bound to the active site of RmlC or RmlD. The Zn2+ ion and residues of its co-ordination sphere are shown in green and orange respectively. Black arrows indicate structural motifs not present in the cupin domain of CapF. Tyr128 (blue) and NADPH (yellow) of RmlD, and Tyr103 (orange) of the SDR domain of CapF are depicted in (B). The curved arrow suggests a mechanism of closure of Loop55–72 of CapF. The co-ordinates of RmlC and RmlD were retrieved from the PDB under accession codes 2IXH and 1KC3 respectively.

RmlD and the N-terminal domain of CapF displayed homologous three-dimensional structures (RMSD=2.4 Å over 189 residues) and both enzymes required the coenzyme NADPH for catalysis (Figure 9B). NADPH and the substrate analogue of RmlD (dTDP-L-rhamnose) fitted well on to the binding pocket of CapF upon superposition of both structures. We found severe bumps (distances below 2.2 Å) at only four residues of CapF in the binding configuration shown in Figure 9(B): the substrate analogue bumped into Asn132 and Asn162, whereas NADPH bumped into Arg33 and Pro59. This rough model thus could recapitulate the approximate orientation of coenzyme and intermediate 3 when bound to CapF.

The biggest difference between the aligned residues of CapF and RmlD occurred in Loop55–72 of CapF, which corresponded to Loop63–84 of RmlD (Figure 9B). Loop55–72 of CapF is largely shifted towards the solvent compared with the equivalent region of RmlD (Figure 9B). In RmlD this loop stabilizes the binding of NADPH through several hydrogen bonds (distance≤3.0 Å) with Ala63 and Thr65 (not shown). In contrast, Loop55–72 of CapF bumps with the adenosine moiety of NADPH in this configuration. Intriguingly, we have shown above that a large portion of Loop55–72 of CapF was the most dynamic region within the crystal structure of CapF (Figures 3 and 4).

On the basis of this comparison, we suggest that Loop55–72 of CapF closes towards the core of the enzyme upon binding of the coenzyme and/or substrate. All of this evidence suggested that the structure of apoCapF described in the present study corresponded to an ‘open’ form in the SDR domain. A similar mechanism to that proposed in the present study has been documented in another homologue SDR protein known as RmlB (Supplementary Table S2). Loop82–102 of RmlB swings between an open and a closed form upon binding of the substrate [46,51,64]. We expect to see analogous closure movements in forthcoming crystal structures of CapF.

Conclusions

We solved the three-dimensional structure of CapF in two different crystal forms. CapF forms a distinctive homodimer with a unique topology built on two functional domains. Comparative analysis demonstrated that the N-terminal domain belongs to the SDR family of proteins, whereas the C-terminal domain is homologous to the cupin motif and displays a bound Zn2+ ion. Each domain possesses a functional role in the reactions catalysed by CapF. The cupin domain is necessary for the C3-epimerization reaction and does not require coenzyme. Meanwhile, the N-terminal domain catalyses NADPH-dependent reduction of the UDP-4-hexulose intermediate. Because the crystal structures corresponded to an open form of the SDR domain, we proposed that CapF experiences a large reconfiguration of its active site during each catalytic cycle. In fact, binding and release of coenzyme from the SDR domain depended on the thermodynamic properties of the interaction. Looking ahead, we will need to address why CapF of S. aureus evolved this unique enzyme containing both an epimerase and a reductase domain within a single polypeptide chain.

Abbreviations

     
  • CP

    capsular polysaccharide

  •  
  • ITC

    isothermal titration calorimetry

  •  
  • RMSD

    root mean square deviation

  •  
  • SDR

    short-chain dehydrogenase/reductase

  •  
  • SeMet

    selenomethionine

  •  
  • UDP-L-FucNAc

    uridine diphosphate N-acetyl-L-fucosamine

  •  
  • UDP-D-GlcNAc

    uridine diphosphate N-acetyl-D-glucosamine

AUTHOR CONTRIBUTION

Takamitsu Miyafusa designed the research, performed experiments, analysed data and edited the manuscript prior to submission. Jose Caaveiro designed the research, performed experiments, analysed data and wrote the paper. Yoshikazu Tanaka designed research, performed experiments and analysed data. Kouhei Tsumoto designed the overall study, analysed data, provided guidance and edited the paper prior to submission.

We thank members of Photon Factory (Tsukuba, Japan) for their assistance during X-ray data collection. We thank Professor M. Kuroda (Center for Pathogen Genomics, National Institute of Infectious Diseases, Tsukuba University) and Professor T. Ohta (Department of Microbiology, Tsukuba University) for providing plasmids containing full-length CapF and CapE. We also appreciate useful suggestions from Professor M. Yao and Professor I. Tanaka (both at the Division of Biological Science, Hokkaido University).

FUNDING

This work was supported in part by a Grant-in-Aid for General Research from the Japan Society for the Promotion of Science [grant number 21360398 (to K.T.)]. T.M. was supported by a predoctoral fellowship from the Japan Society for the Promotion of Science [grant number 09J07066].

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Author notes

The atomic co-ordinates and the structure factors of CapF crystallized in space group P3221 and space group C2221 will appear in the PDB under accession codes 3ST7 and 3VHR respectively.

1

Present address: Creative Research Initiative Sousei, Hokkaido University, Sapporo 001-0021, Japan.

Supplementary data