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

• Published in: Structure (London) 2011-12, Vol.19 (12), p.1773-1783
• Main Authors: Petkun, Svetlana, Shi, Rong, Li, Yunge, Asinas, Abdalin, Munger, Christine, Zhang, Linhua, Waclawek, Mandy, Soboh, Basem, Sawers, R. Gary, Cygler, Miroslaw
• Format: Article
• Language: English
• Subjects: Acid Anhydride Hydrolases - chemistry; Acid Anhydride Hydrolases - metabolism; Acylphosphatase; Binding Sites; Carbamyl Phosphate - metabolism; Carboxyl and Carbamoyl Transferases - chemistry; Carboxyl and Carbamoyl Transferases - metabolism; Catalysis; Catalytic Domain; Escherichia coli - enzymology; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - metabolism; Hydrogenase - chemistry; Ligands
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2011.09.023

[NiFe]-hydrogenases are multimeric proteins. The large subunit contains the NiFe(CN)2CO bimetallic active center and the small subunit contains Fe-S clusters. Biosynthesis and assembly of the NiFe(CN)2CO 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 Zn2+ 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.