Molecular characterization and heterologous expression of hypCD, the firsttwo [NiFe] hydrogenase accessory genes of Thermococcus litoralis

The hypCD genes, encoding the counterparts of mesophilic proteins involved in the maturation of [NiFe] hydrogenases, were isolated from the hypertherm ophilic archaeon Thermococcus litoralis. The deduced gene products showed 3 0-40% identity to the corresponding mesophilic proteins. HypC and HypD were synthesized by the T7 expression system. Heterologous complementation expe riments were done in Escherichia coli and Ralstonia eutropha strains lackin g functionally active hypC and hypD genes. Only the cytoplasmic hydrogenase of R. eutropha could be processed by HypD from T litoralis. This was the f irst demonstration of mesophilic hydrogenase processing using a hyperthermo philic archaeal accessory protein to produce an active enzyme.