Authors: Blokesch, Melanie; Paschos, Athanasios; Bauer, Anette; Reissmann, Stefanie; Drapal, Nikola; Böck, August

Source: FEBS Journal, Volume 271, Number 16, August 2004 , pp. 3428-3436(9)

Publisher: Wiley-Blackwell

Abstract:

The hydrogenase maturation proteins HypF and HypE catalyze the synthesis of the CN ligands of the active site iron of the NiFe-hydrogenases using carbamoylphosphate as a substrate. HypE protein from Escherichia coli was purified from a transformant overexpressing the hypE gene from a plasmid. Purified HypE in gel filtration experiments behaves predominantly as a monomer. It does not contain statistically significant amounts of metals or of cofactors absorbing in the UV and visible light range. The protein displays low intrinsic ATPase activity with ADP and phosphate as the products, the apparent K_m being 25 µmand the k_cat 1.7 × 10⁻³ s⁻¹. Removal of the C-terminal cysteine residue of HypE which accepts the carbamoyl moiety from HypF affected the K_m (47 µm) but not significantly the k_cat (2.1 × 10⁻³ s⁻¹). During the carbamoyltransfer reaction, HypE and HypF enter a complex which is rather tight at stoichiometric ratios of the two proteins. A mutant HypE variant was generated by amino acid replacements in the nucleoside triphosphate binding region, which showed no intrinsic ATPase activity. The variant was active as an acceptor in the transcarbamoylation reaction but did not dehydrate the thiocarboxamide to the thiocyanate. The results obtained with the HypE variants and also with mutant HypF forms are integrated to explain the complex reaction pattern of protein HypF.

Keywords: NiFe hydrogenase; maturation; CN ligand synthesis; hypE mutations; carbamoyl transfer

Document Type: Research article

DOI: http://dx.doi.org/10.1111/j.1432-1033.2004.04280.x

Publication date: 2004-08-01

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• In this Subject: Anatomy & Physiology ,  Biology ,  Chemistry (General) ,  Biochemistry
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