RESUMO
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.
Assuntos
Proteínas Arqueais/genética , Endopeptidases/genética , Hidrogenase/metabolismo , Processamento de Proteína Pós-Traducional , Thermococcus/genética , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Endopeptidases/metabolismo , Hidrogenase/química , Hidrogenase/genética , Multimerização Proteica , Proteólise , Thermococcus/enzimologiaRESUMO
HypB (metal-binding GTPase) and HypA (nickel metallochaperone) are required for nickel insertion into [NiFe] hydrogenase. However, the HypB homolog proteins are not found in some archaeal species including Thermococcales. In this article, we identify a novel archaeal Mrp/MinD family ATPase-type HypB from Thermococcus kodakarensis (Tk-mmHypB) and determine its crystal structure. The mmhypB gene is conserved among species lacking the hypB gene and is located adjacent to the hypA gene on their genome. Deletion of the mmhypB gene leads to a significant reduction in hydrogen-dependent growth of T. kodakarensis, which is restored by nickel supplementation. The monomer structure of Tk-mmHypB is similar to those of the Mrp/MinD family ATPases. The ADP molecules are tightly bound to the protein. Isothermal titration calorimetry shows that Tk-mmHypB binds ATP with a K(d) value of 84 nM. ADP binds more tightly than does ATP, with a K(d) value of 15 nM. The closed Tk-mmHypB dimer in the crystallographic asymmetric unit is consistent with the ATP-hydrolysis-deficient dimer of the Mrp/MinD family Soj/MinD proteins. Structural comparisons with these proteins suggest the ATP-binding dependent conformational change and rearrangement of the Tk-mmHypB dimer. These observations imply that the nickel insertion process during the [NiFe] hydrogenase maturation is performed by HypA, mmHypB, and a nucleotide exchange factor in these archaea.
