Abstract
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.
Highlights
Hydrogen metabolism in enterobacteria involves the activity of the products of three functional classes of genes that code for structural proteins, regulatory proteins, or for proteins involved in metal center biosynthesis and enzyme maturation
The synthesis of the CO and CN ligands of [NiFe] hydrogenases and their attachment to the iron poses a number of intriguingly novel features of bioinorganic chemistry
It is still unclear whether they are attached to the iron when it has been inserted into the large hydrogenase subunit or whether they are preformed, e.g. at some scaffold protein and transferred as the complete entity into the apoprotein
Summary
E. coli Strains, Plasmids, and Growth Conditions—Strain MC4100 [19] from E. coli was used as wild type, and strains JM109 [20] and DH5␣ [21] served as hosts for transformations. Cell extracts or purified HypF proteins were incubated in 1-ml reaction mixtures containing 50 mM Tris/Cl, pH 7.4, 100 mM KCl, 1 mM MgCl2, 0.1 mM DTT, and the CP concentration indicated at 30 °C. Dependence of ATP Hydrolysis by HypF Protein on the Presence of Carbamoyl Phosphate—ATP cleavage by HypF was measured in 1-ml reaction volumes containing 50 mM Tris/Cl, pH 7.4, 100 mM KCl, 1 mM MgCl2, 0.1 mM DTT, 25 nM HypF, 50 g of bovine serum albumin per ml, and [␥-32P]ATP (specific radioactivity of 16.1 Ci/mol) and carbamoyl phosphate at the indicated concentrations. [32P]PPi-ATP Exchange Assay—PPi-ATP exchange activity catalyzed by HypF was determined in 1-ml assays containing 50 mM Tris/Cl, pH 7.4, 100 mM KCl, 1 mM MgCl2, 0.1 mM DTT, 0.1 mM ATP, 0.1 mM [32P]PPi (specific radioactivity 25 Ci/mol), carbamoyl phosphate at the indicated concentrations, and 62.5 nM HypF protein. It was purchased from Sigma or from ICN Biomedical Inc. (Eschwege, Germany). [␣-32P]ATP, [␥-32P]ATP, and labeled inorganic pyrophosphate were purchased from PerkinElmer Life Sciences
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