Structural basis for the reaction mechanism of S-carbamoylation of HypE by HypF in the maturation of [NiFe]-hydrogenases

Abstract

As a remarkable structural feature of hydrogenase active sites, [NiFe]-hydrogenases harbor one carbonyl and two cyano ligands, where HypE and HypF are involved in the biosynthesis of the nitrile group as a precursor of the cyano groups. HypF catalyzes S-carbamoylation of the C-terminal cysteine of HypE via three steps using carbamoylphosphate and ATP, producing two unstable intermediates: carbamate and carbamoyladenylate. Although the crystal structures of intact HypE homodimers and partial HypF have been reported, it remains unclear how the consecutive reactions occur without the loss of unstable intermediates during the proposed reaction scheme. Here we report the crystal structures of full-length HypF both alone and in complex with HypE at resolutions of 2.0 and 2.6 Å, respectively. Three catalytic sites of the structures of the HypF nucleotide- and phosphate-bound forms have been identified, with each site connected via channels inside the protein. This finding suggests that the first two consecutive reactions occur without the release of carbamate or carbamoyladenylate from the enzyme. The structure of HypF in complex with HypE revealed that HypF can associate with HypE without disturbing its homodimeric interaction and that the binding manner allows the C-terminal Cys-351 of HypE to access the S-carbamoylation active site in HypF, suggesting that the third step can also proceed without the release of carbamoyladenylate. A comparison of the structure of HypF with the recently reported structures of O-carbamoyltransferase revealed different reaction mechanisms for carbamoyladenylate synthesis and a similar reaction mechanism for carbamoyltransfer to produce the carbamoyl-HypE molecule.

FIGURE 1.

Functional and structural aspects of HypF. A, reaction scheme of carbamoyltransfer from carbamoylphosphate to the C-terminal cysteine of HypE catalyzed by HypF. B, overall structure of HypF from C. subterraneus. The protein molecule is schematically represented using a different color for each domain. The phosphate ion, AMPCPP, and AMMPCP molecules are drawn as stick models, and the zinc, magnesium, and iron atoms are depicted by gray, cyan, and brown spheres, respectively. The long channel connecting the phosphate-binding site with the nucleotide-binding site in the YrdC-like domain is indicated by a light blue surface model. One of the two zincs is hidden behind the surface representation. The N and C termini of the polypeptide are labeled.

FIGURE 2.

Phosphate-binding site in the ACP domain of the full-length HypF. The ACP and Zn finger-like domains are illustrated in cyan and green, respectively. A and B provide the same view of the same molecule, highlighting different contacts. W1–4 represents defined water molecules in the crystal structure. Dotted gray lines represent hydrogen bonds. A, direct contacts to the bound-phosphate ion and proposed catalytic water molecule. B, contacts by residues in the Zn finger-like domain.

FIGURE 3.

Nucleotide-binding sites of HypF from C. subterraneus in complex with nucleotides. The bound nucleotides are shown as stick models. Iron and magnesium ions and water molecules are depicted with balls of brown, cyan, and red, respectively. Hydrogen and coordination bonds are depicted by dashed lines of gray and pink, respectively. The F_o-F_c electron density maps around the bound nucleotides contoured at 3.0 σ are represented by blue meshes, where non-protein atoms were omitted in the calculation of F_c. For clarity, interactions with the adenosine moieties are not shown. A, nucleotide-binding site in the YrdC-like domain. B, nucleotide-binding site in the Kae1-like domain.

FIGURE 4.

UV-VIS absorption spectra of HypF. The spectrum was measured for 20 μm protein in the buffer containing 50 mm HEPES (pH 7.4) and 100 mm NaCl for HypF alone (green line). The dithiothreitol-reduced form (blue line) and the dithionite-reduced form (red line) were prepared by adding 10 mm dithiothreitol and 10 mm dithionite, respectively, and then incubated for 10 min at room temperature.

FIGURE 5.

Structure of the HypE-HypF complex. A, overall structure of HypE-HypF heterotetramer. The HypE molecules are shown with different colors for each protomer. The N- and C-terminal domains of HypE are labeled, and the magnesium ions found at the boundary of two protomers are represented by green spheres. A pseudo dyad is indicated at the center of the top figure. The same color scheme used in Fig. 1B depicts the HypF molecules. B, superposition of overall structures of HypE homodimers, corresponding to the black square region in the top of A. Structures shown are HypE of C. subterraneus (green; PDB ID), D. vulgaris (blue; PDB ID: 2Z1U), T. kodakarensis (magenta; PDB ID: 2Z1E), and E. coli (yellow; PDB ID:2RB9). The N termini of C. subterraneus and D. vulgaris orthologs are labeled. The 2-fold axis of the homodimers is shown at the center of the enlarged view. The loop region involved in the segment-swapping observed in D. vulgaris and E. coli orthologs is encircled with the dotted red line. C, close-up view of the interface between HypE and HypF, corresponding to the black square region in the bottom of A. The residues involved in the subunit interaction are depicted as stick models, where the major and minor interaction sites are enclosed by red and blue dotted lines, respectively. Four residues coordinating to the iron atom (brown sphere) and Pro-342 are also shown with stick models.

FIGURE 6.

Comparison of HypF structures of HypF alone and HypE-HypF complex. Two structures were superposed by using the Cα atoms in the YrdC-like and Kae1-like domains. HypF alone is shown with the same color scheme used in Fig. 1B whereas HypF in the HypE-HypF complex is shown in gray throughout the entire polypeptide. For clarity, the ACP domains are represented by cartoon models. The dislocation of the ACP domain can be simplified by two rotational movements (1 and 2).

FIGURE 7.

Schematic drawing of the reaction mechanism of the three consecutive reactions catalyzed by HypF. The first step is hydrolysis of carbamoylphosphate, where the water molecule (red) is activated for nucleophilic attack on the phosphate group to release carbamate (blue), which is transferred to the nucleotide-binding site in the YrdC-like domain via the channel identified in the crystal structure of HypF alone. The second step includes nucleophilic attack of carbamate on the α-phosphate group of ATP to form the carbamoyladenylate intermediate, where the protein merely provides residues for maintaining carbamate in a suitable position for the reaction. The carbamoyladenylate is then translocated to the nucleotide-binding site in the Kae1-like domain, enhanced by the change in charge properties of the molecule because of the release of the pyrophosphate group as suggested in previous studies (22, 49). The last step occurs at the iron-binding site in the Kae1-like domain. The conserved Glu-617 (not shown in the figure) may play a role in proton transfer during the reaction, but the exact mechanism is still unknown. One of the hydrogen-bond acceptors is missing for all amine groups of the substrates, suggesting that a minor conformational rearrangement and/or binding of solvent molecules are required to catalyze each reaction.