CHARACTERIZATION OF A PROTEASE FROM ESCHERICHIA-COLI INVOLVED IN HYDROGENASE MATURATION

The large subunits of nickel-containing hydrogenases are synthesised i n a precursor form which? after nickel incorporation, is processed by proteolytic cleavage at the C-terminal end. The protease involved in p rocessing of HycE, the large subunit of hydrogenase 3 from Escherichia coli, was purified by three chromatographic steps to apparent homogen eity. Its gene was identified by using a hybridisation probe generated by PCR with oligonucleotide primers the sequence of which was derived from the N-terminal and internal amino acid sequences. Determination of the nucleotide sequence showed that the gene is located distally an d as a hitherto uncharacterised gene within the hyc operon, coding for hydrogenase 3 components. It was designated hycI. The HycI protease h as a molecular mass of 17 k Da and is a monomer. Its cleavage reaction is not inhibited by conventional inhibitors of serine and metalloprot eases, which correlates with the fact that the sequence does not conta in signature motifs characteristic of serine-, metallo-, cysteine- or acid proteases. Homologous genes are present in other transcriptional units coding for hydrogenases.