HypF, a carbamoyl phosphate-converting enzyme involved in [NiFe] hydrogenase maturation.
HypF has been characterized as an auxiliary protein whose function is required for the synthesis of active [NiFe] hydrogenases in Escherichia coli and other bacteria. To approach the functional analysis, in particular the involvement in CO/CN ligand synthesis, HypF was purified from an overproducing strain to apparent homogeneity. The purified protein behaves as a monomer on size exclusion chromatography, and it is devoid of nickel or other cofactors. As indicated by the existence of a sequence motif also present in several O-carbamoyltransferases, HypF interacts with carbamoyl phosphate as a substrate and releases inorganic phosphate. In addition, HypF also possesses ATP cleavage activity that gives rise to AMP and pyrophosphate as products and that is dependent on the presence of carbamoyl phosphate. This and the fact that HypF catalyzes a carbamoyl phosphate-dependent pyrophosphate ATP exchange reaction suggest that the protein catalyzes activation of carbamoyl phosphate. Extensive mutagenesis of the putative functional motifs deduced from the derived amino acid sequence showed a full correlation of the resulting variants between their activity in hydrogenase maturation and the in vitro reactivity with carbamoyl phosphate. The results are discussed in terms of the involvement of HypF in the conversion of carbamoyl phosphate to the CN ligand.
Paschos
Athanasios
A
Mikrobiologie, Department I der Fakultät für Biologie, Ludwig-Maximilians-Universität München, Maria-Ward-Strasse 1a, D-80638 Munich, Germany.
Bauer
Anette
A
Zimmermann
Anja
A
Zehelein
Eva
E
Böck
August
A
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000502:physiology*; D002221:Carbamyl Phosphate / Q000378:metabolism; D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D006864:Hydrogenase / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008024:Ligands; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D001426:Bacterial Proteins; C108878:HypF protein, Bacteria; D008024:Ligands; D000249:Adenosine Monophosphate; D002221:Carbamyl Phosphate; D000255:Adenosine Triphosphate; C043296:nickel-iron hydrogenase; D006864:Hydrogenase