A novel very small subunit of a selenium containing [NiFe] hydrogenease of Methanococcus voltae is postranslationally processed by cleavage at a defined position

Abstract

A c~enzyme-F~~,, non-reducing [NiFe] hydrogenase was isolated from Methanococcus voltae. It consists of three subunits. They are the products of the previously identified genes vhuA, vhuG and vhuU. The vhuU gene product is of only 25 amino acids. This novel very small hydrogenase subunit contains selenocysteine within a conserved amino-acid sequence previously shown to be involved in Ni coordination. The subunit is shorter than the predicted primary gene product and is therefore apparently post-translationally processed.[NiFe] hydrogenases are widespread in prokaryotes. At least their functional domains, carrying Fe-S clusters or the nickel-containing reaction center are believed to be descendants of a common prototype since strong similarities can be seen in all known enzymes of this type [l, [4]. One of the enzymes (the fruA,G,B product), which is able to reduce the deazaflavine Fdz0, has previously been purified [5]. It contains selenium. In the gene of the largest subunit (fruA) a single internal TGA codon is found in the region coding for the assumed Ni-binding site. It most likely encodes selenocysteine. This notion is supported by the fact that the TGA codon is followed by a sequence capable of forming a moderately stable secondary structure in the transcript. These two features are known to be prerequisites for the incorporation of selenocysteine into formate dehydrogenase in Escherichia coli [6].A second hydrogenase gene group (vhuG,A, U) has been predicted to encode a selenium containing F420 non-reducing hydrogenase. Again a TGA codon and an adjacent downstream sequence capable of secondary-structure formation are seen in the homologous position to the one of the previously mentioned hydrogenase [4]. The DNA sequence analysis led to the prediction of a very small subunit containing the selenocysteine, which would carry at least part of the Ni-coordination site, whereas the homologous domain would be part of the largest subunit of the putative selenium-free vhc hydrogenase (Fig. 1). Such a small subunit has never been found in other [NiFe] hydrogenases and might be a very useful tool for the study of the primary reaction center of the enzyme.In this study we describe this new [NiFe] hydrogenase from M. voltae which does comprise three subunits. One of them is indeed very small and contains selenocysteine. To our surprise the primary translation product of the small gene appears to be processed. Removal of a predicted C-terminal portion of 44 amino-acid residues leads to the final length of only 25 amino acids and a molecular mass of only 2884 Da if the N-terminal methionine is removed. EXPERIMENTAL PROCEDURES Cell growth and enyzme isolationM. voltae cells were grown to late exponential phase as described previously [7] in a 300-1 fermenter. After harvesting under aerobic conditions they were frozen in liquid nitrogen. All enzyme purification steps were performed under anaerobic conditions. Crude lysate and centrifugation10 g cells were lysed in 30 ml buffer A (50 mM TrisMCl, pH 7.5) containing 10...

Requirement for nickel of the transmembrane translocation of NiFe‐hydrogenase 2 in Escherichia coli

“…In the purified coenzyme-F420 non-reducing selenium-containing NiFe-hydrogenase from Methanococcus voltae, this conserved motif is located on a third subunit composed of 25 residues. The corresponding vhuU gene encodes a precursor of 44 amino acids whose C-terminal part is removed after the histidyl residue during the processing [12]. The structural data indicate that the two conserved cysteines in the motif DPCxxCxxH in the large subunits of D. gigas provide the ligands required for the binding of nickel [11].…”

Cloning, molecular analysis and insertional mutagenesis of the bidirectional hydrogenase genes from the cyanobacterium Anacystis nidulans

“…Near the carboxy terminus, HoxH possesses a strongly conserved motif ending with a histidine. The formation of the mature protein and the incorporation of Ni into it might require the cleavage of a stretch of 26 amino acids behind this His similar as in the proteins from Methanococcus voltae [16], E. eoli (hydrogenase 3) [17], and A. eutrophus [18]. An N-terminal leader sequence is also not observed in HoxH of A. nidulans.…”

Fusion of two subunits does not impair the function of a [NiFeSe]‐hydrogenase in the archaeon Methanococcus voltae

“…6). In the case of the wild-type enzyme, the 46-kDa VhuA subunit [14] was detected. In the case of the mutant enzyme, the antiserum reacted with a larger polypeptide.…”

“…Therefore, this strain does not carry genetic information for the wild-type Vhu-hydrogenase. The purification of the Vhuhydrogenase and the mutant enzyme was performed as described [14] with the following modifications. The active DEAE-Sepharose fraction was adjusted to 0.8 M ammonium sulfate in 50 mM Tris/HCl pH 7.5 (buffer A).…”

“…It appears that the gene normally encoding the largest subunit has been split. The homologue of the larger N-terminal part of the subunit carrying the primary reaction center is encoded by the gene vhuA, directly followed by a very small gene vhuU encoding what would be the C-terminal part of a normal large subunit [14,17]. The vhuU gene product is C-terminally processed, probably prior to its incorporation into the enzyme and the formation of the [NiFe] cluster to which it contributes a cysteinyl residue and a selenocysteinyl residue.…”