Athanasios Paschos

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.

Carbamoylphosphate requirement for synthesis of the active center of (NiFe)-hydrogenases

The iron of the binuclear active center of [NiFe]‐hydrogenases carries two CN and one CO ligands which are thought to confer to the metal a low oxidation and/or spin state essential for activity. Based on the observation that one of the seven auxiliary proteins required for the synthesis and insertion of the [NiFe] cluster contains a sequence motif characteristic of O‐carbamoyl‐transferases it was discovered that carbamoyl phosphate is essential for formation of active [NiFe]‐hydrogenases in vivo and is specifically required for metal center synthesis suggesting that it is the source of the CO and CN ligands. A chemical path for conversion of a carbamoyl group into cyano and carbonyl moieties is postulated

Analysis of the cleavage site specificity of the endopeptidase involved in the maturation of the large subunit of hydrogenase 3 from Escherichia coli.

Abstract The maturation of [NiFe]-hydrogenases is a catalysed process in which the activities of at least seven proteins are involved. The last step consists of the endoproteolytic cleavage of the precursor of the large subunit after the [NiFe]-metal centre has been assembled. The amino acid sequence requirements for the endopeptidase HycI involved in the C-terminal processing of HycE, the large subunit of the hydrogenase 3 from Escherichia coli, were investigated. Mutational alteration of the amino acid residues neighbouring the cleavage site showed that proteolysis still occurred when chemically similar amino acids were exchanged. Processing was blocked, however, in a variant in which the methionine at the C-terminal side was replaced by a glutamate residue. Truncation of the precursor from the C-terminal end rendered variants amenable to maturation even when two-thirds of the extension were removed but abolished proteolysis upon further deletion of a cluster of six basic amino acids. A construct in which the C-terminal extension from the large subunit of the hydrogenase 2 was fused to the mature part of the large subunit of hydrogenase 3 was neither processed by HycI nor by HybD, the endopeptidase specific for the large subunit of hydrogenase 2. The maturation endopeptidase, therefore, exhibits a relaxed sequence constraint in recognition of its cleavage site and does not require the entire C-terminal extension. The results point to an interaction of the C-terminus with some domain of the large subunit, rendering a conformation amenable to recognition by the endopeptidase.

Generation of the [NiFe] Hydrogenase Active Site (Sulfur's the One!)

Abstract Chemical and biochemical methods were used to unravel the unprecedented pathway by which the CN ligands of iron in [NiFe] hydrogenase are introduced. Carbamoyl phosphate is the one carbon precursor of these ligands, and reactions involving a protein cysteinyl sulfur are key for processing this precursor into CN ligands.

Hydrogenase maturation endopeptidase

Publisher Summary This chapter elaborates the structural chemistry and the biological aspects of hydrogenase maturation endopeptidase. [NiFe]Hydrogenases consist of subunits of 30–35 kDa and of 60–65 kDa, depending on the organism. The large subunit carries the [NiFe]metallocenter, and it is synthesized in a precursor form and proteolytically processed to the form present in the mature enzyme. Hycl and HybD endopeptidases are monomeric proteins with molecular masses of 17 kDa and 17.5 kDa, respectively. There is no primary structure motifs characteristic of serine, cysteine or metalloproteases. Hycl lacks the N-terminal methionine. The crystal structure of HybD, which cleaves off 15 amino acid peptide from the C-terminus of the precursor of the large subunit of hydrogenase 2 from E. coli, has been solved at 2.2 A resolutions. HybD crystallizes with a cadmium ion from the crystallization buffer whereby the metal is pentacoordinated by Glul6, Asp62 and His93, and a water molecule. These residues are strictly conserved amongst other members of the hydrogenase maturation endopeptidase family. Their replacement either abolishes processing activity completely or it impairs activity. The function of Hycl and HybD endopeptidases is the formation of active hydrogenase 3 and 2, respectively, in E. coli.