Structure of Hydrogenase Maturation Protein HypF with Reaction Intermediates Shows Two Active Sites

Abstract

[NiFe]-hydrogenases are multimeric proteins. The large subunit contains the NiFe(CN)(2)CO bimetallic active center and the small subunit contains Fe-S clusters. Biosynthesis and assembly of the NiFe(CN)(2)CO active center requires six Hyp accessory proteins. The synthesis of the CN(-) ligands is catalyzed by the combined actions of HypF and HypE using carbamoylphosphate as a substrate. We report the structure of Escherichia coli HypF(92-750) lacking the N-terminal acylphosphatase domain. HypF(92-750) comprises the novel Zn-finger domain, the nucleotide-binding YrdC-like domain, and the Kae1-like universal domain, also binding a nucleotide and a Zn(2+) ion. The two nucleotide-binding sites are sequestered in an internal cavity, facing each other and separated by ∼14 Å. The YrdC-like domain converts carbamoyl moiety to a carbamoyl adenylate intermediate, which is channeled to the Kae1-like domain. Mutations within either nucleotide-binding site compromise hydrogenase maturation but do not affect the carbamoylphosphate phosphatase activity.

NMR-based Structural Analysis of Threonylcarbamoyl-AMP Synthase and Its Substrate Interactions

“…3). The structure is consistent with all protein structures solved to date in the TsaC/Sua5 family, such as YciO (28), Sua5 (29), and HypF (30). They all have an ␣/␤ twisted open-sheet topology with parallel and antiparallel adjacent ␤-strands and a central concave cavity lined with positive electrostatic potential.…”

“…These residues have been shown to surround the adenine-binding site in co-crystal structures of both E. coli HypF (middle domain, residues 188 -378) and S. tokodaii Sua5 with the nonhydrolysable ATP analog AMP-PNP (30,50). For HypF, the adenine is buried in a hydrophobic environment between Leu-277 and Pro-249, and it forms weak hydrogen bonds with Glu-296 and Arg-372 (30). Pro-249 near the ATP-binding site in HypF is not conserved in this family of proteins and is not present in TsaC.…”

“…Within the TsaC/Sua5 family, four structures have been solved by x-ray crystallography, including E. coli YciO (28), Sulfolobus tokodaii Sua5 (29), E. coli HypF (30), and E. coli TsaC (27). Each of these structures contains a TsaC domain with a unique folding pattern and the ability to catalyze similar chemistries.…”

Structural Basis for the Reaction Mechanism of S-Carbamoylation of HypE by HypF in the Maturation of [NiFe]-Hydrogenases

“…HypF is generally composed of four domains: the acylphosphatase (ACP) 3 domain, Zn finger-like domain, YrdC-like domain, and Kae1-like domain (17) (Fig. 1B).…”

“…The exact reaction that takes place at the Kae1-like domain, which contains another nucleotide-binding site and a mononuclear iron/zinc site has not been elucidated. While crystal structures of HypF from Escherichia coli have been reported for the N-terminal ACP domain (residues 1-91) (18) and for the rest of the C-terminal region (residues 92-750) (17), the intact structure of HypF is not yet available. On the other hand, the crystal structures of the full-length HypE from three different organisms have already been reported (12,19,20).…”