No abstract

The maturation of [NiFe]-hydrogenases requires a number of accessory proteins, which include hydrogenase-specific endopeptidases. The endopeptidases carry out the final cleavage reaction of the C-terminal regions of [NiFe]-hydrogenase large subunit precursors. The hyperthermophilic archaeon Thermococcus kodakarensis harbors two [NiFe]-hydrogenases, a cytoplasmic Hyh and a membrane-bound Mbh, along with two putative hydrogenase-specific endopeptidase genes. In this study, we carried out a genetic examination on the two endopeptidase genes, TK2004 and TK2066. Disruption of TK2004 resulted in a strain that could not grow under conditions requiring hydrogen evolution. The Mbh large subunit precursor (pre-MbhL) in this strain was not processed at all whereas Hyh cleavage was not affected. On the other hand, disruption of TK2066 did not affect the growth of T. kodakarensis under the conditions examined. Cleavage of the Hyh large subunit precursor (pre-HyhL) was impaired, but could be observed to some extent. In a strain lacking both TK2004 and TK2066, cleavage of pre-HyhL could not be observed. Our results indicate that pre-MbhL cleavage is carried out solely by the endopeptidase encoded by TK2004. Pre-HyhL cleavage is mainly carried out by TK2066, but TK2004 can also play a minor role in this cleavage.

[NiFe] hydrogenase catalyzes the reversible oxidation of molecular hydrogen. The large subunit of the enzyme carries a NiFe(CN) 2 (CO) cluster at the active site. The biosynthesis of the NiFe cluster needs maturation proteins, HypA, HypB, HypC, HypD, HypE, and HypF. After incoorporation of the Fe(CN) 2 CO group, HypB inserts the Ni atom together with HypA. Previously charactrized HypB protein belongs to the G3E family GTPase and GTP hydrolysis is required for the Ni insertion process and maturation. A gene encoding the G3E family HypB is not found in genome of some archaea. On the other hand, a gene showing high sequence similarity to the Mrp/MinD family ATPase is conserved adjacent to the hypA gene on their genome, assuming that this gene encodes a functional homologue of HypB. Here, we identify TK2007 gene product from hyperthermophilic archaeon Thermococcus kodakarensis KOD1 as HypB and determine its crystal structure. A severe growth defect of the ÄTK2007 strain under hydrogenase-required condition was observed and restored by addition of Ni ions, providing convincing evidence that the TK2007 gene product is a novel Mrp/MinD family HypB protein in T. kodakarensis (Tk-mmHypB). The structure of Tk-mmHypB was solved as homodimer related by a non-crystallographic 2-fold axis. Each monomer consists of a central seven-stranded parallel b-sheet surrounded by a-helices. Intriguingly, ADP molecules from E. coli expression system are tightly bound to the protein although we did not attempted to co-crystallize the purified sample with an ATP, suggesting that Tk-mmHypB shows high affinity with an ADP molecule. Significant structural differences between monomers indicate that the C-terminal loop-helix-loop region is related to the affinity for the ADP. Furthermore, the structure around the dimer interface reveals that the dimer formation is required for ATP hydrolysis.Comparison of the nucleotide-binding site with that of the Mrp/MinD family nitrogenase iron NifH protein suggests structural change during the hydrolysis. These structural insights imply the Ni insertion mechanism depending on nucleotide-exchange factor. Our studies on Tk-mmHypB shed new light on structural and functional diversity of HypB proteins in the Ni insertion process in the [NiFe] hydrogenase maturation.