Fusion of two subunits does not impair the function of a [NiFeSe]-hydrogenase in the archaeon Methanococcus voltae.
[NiFe]-hydrogenases generally carry the bimetallic Ni-Fe reaction center on their largest subunit. The [NiFeSe]-hydrogenase Vhu from Methanococcus voltae has an unusual subunit composition. Some of the amino acids participating in the formation of the reaction center are within a separate, very small subunit, called VhuU. It consists of only 25 amino acids and contains the selenocysteinyl residue, a ligand to the Ni atom. We have tested whether the special configuration of the Vhu-hydrogenase is of particular biochemical relevance. We have constructed a fusion subunit derived from the VhuA and VhuU subunits by generating a gene fusion which was inserted into the chromosome of M. voltae by gene replacement. The enzyme was purified and shown to be as active as the wild-type enzyme. M. voltae carries the genetic information for four different [NiFe]-hydrogenases. In addition to the Vhu-hydrogenase, a second selenium-containing enzyme, Fru, has been purified. Two selenium-free enzymes, Vhc and Frc, are homologues of Vhu and Fru, respectively. Their gene groups, vhc and frc are transcribed only upon selenium depletion. The selenium-containing subunit VhuU has been implicated in their negative regulation. However, cells containing the fusion hydrogenase still exhibited normal regulation of the vhc andfrc promoter activities as tested in reporter gene constructs. This indicates that the free VhuU polypeptide is not required for the negative regulation of the vhc or frc genes.
Genetics, Department of Biology, University of Marburg, Germany.
M
M
Pfeiffer
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R
Bingemann
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A
Klein
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D006864:Hydrogenase / Q000737:chemistry*; D007527:Isoenzymes / Q000235:genetics; D008667:Metalloproteins / Q000737:chemistry; D017017:Methanococcus / Q000201:enzymology*; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000502:physiology; D051140:Selenoproteins; D017422:Sequence Analysis, DNA; D014162:Transfection / Q000235:genetics
D001426:Bacterial Proteins; D007527:Isoenzymes; D008667:Metalloproteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D051140:Selenoproteins; C058560:nickel-iron-selenium hydrogenase; D006864:Hydrogenase