tailieunhanh - Báo cáo khoa học: Analysis of the transcarbamoylation-dehydration reaction catalyzed by the hydrogenase maturation proteins HypF and HypE

The hydrogenase maturation proteins HypF and HypE catalyze the synthesis of theCNligands of the active site iron of the NiFe-hydrogenases using carbamoylphosphate as a substrate. HypE protein fromEscherichia coliwas purified from a transformant overexpressing thehypEgene from a plasmid. PurifiedHypE in gel filtration experiments behaves predominantly as a monomer. It does not contain statisti-cally significant amounts ofmetals or of cofactors absorbing in the UV and visible light range. | Eur. J. Biochem. 271 3428-3436 2004 FEBS 2004 doi Analysis of the transcarbamoylation-dehydration reaction catalyzed by the hydrogenase maturation proteins HypF and HypE Melanie Blokesch Athanasios Paschos Anette Bauer Stefanie Reissmann Nikola Drapal and August Bock Department Biologie I Mikrobiologie Ludwig-Maximilians-Universitait MUnchen Miinchen Germany 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 Km being 25 M and the kcat X 10 3 s 1. Removal of the C-terminal cysteine residue of HypE which accepts the carbamoyl moiety from HypF affected the Km 47 M but not significantly the kcat X 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. Escherichia coli possesses four hydrogenases Hyd1-Hyd4 which are all members of the NiFe class 1 2 . In these enzymes the .