Characterisation of a Protease from Escherichia coli Involved in Hydrogenase Maturation

Abstract

The large subunits of nickel-containing hydrogenases are synthesised in a precursor form which, after nickel incorporation, is processed by proteolytic cleavage at the C-terminal end. The protease involved in processing of HycE, the large subunit of hydrogenase 3 from Escherichia coli, was purified by three chromatographic steps to apparent homogeneity. 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 and as a hitherto uncharacterised gene within the hyc operon, coding for hydrogenase 3 components. It was designated hycl. The HycI protease has a molecular mass of 17 k Da and is a monomer. Its cleavage reaction is not inhibited by conventional inhibitors of serine and metalloproteases, which correlates with the fact that the sequence does not contain signature motifs characteristic of serine-, metallo-, cysteine-or acid proteases. Homologous genes are present in other transcriptional units coding for hydrogenases.Keywords. Protease HycI; carboxy-terminal processing ; [NiFeIhydrogenase; Escherichia coli.Escherichia coli possesses three nickel-dependent hydrogenases [l]. Hydrogenase 1 and 2 are uptake hydrogenases involved in anaerobic H, oxidation with the concomitant reduction of an electron acceptor like fumarate; hydrogenase 3 is a part of the formate hydrogenlyase complex which functions in hydrogen evolution during fermentation (for review see [2]). Genes coding for the constituent subunits of these three enzymes are located at three different sites on the E. coli chromosome and are clustered with four (hydrogenase 1 ; hya operon), five (hydrogenase 2; hyb operon) or six (hydrogenase 3; hyc operon) additional genes which code for redox carriers or for proteins of unknown function [3-51. Mutations in most of the hya, hyb and hyc genes from E. coli specifically affect the formation of active hydrogenases 1, 2 and 3, respectively [4, 6, 71. Mutations in other genes (hyp), however, coding outside these three operons block the formation of all three hydrogenases pleiotropically [8, 91, i.e. they appear to have a general function in hydrogenase formation. Possibilities that were considered are folding, metal incorporation or membrane integration. Mutations in several genes of the hya, hyb and hyc operon and inactivation of the hyp genes lead to the accumulation of the large subunits of the hydrogenases in a Cterminally extended form [6,7, 91. This precursor of the large subunit was shown to be present also in several hydrogenase- deficient mutants of other organisms [lo-131 and, in addition, when the cells were starved for nickel [14]. It was amenable to in vitro processing provided that nickel had already been incorporated [15] or that nickel was present in the processing mixture [14]. Processing takes place at a histidine [16-181 or arginine [15] residue three amino acids C-terminal to a cy...

Reassignment of the gene encoding the Escherichia coli hydrogenase 2 small subunit

“…It is likely that the physiological complex includes HybA, HybB, a polypeptide with identity to the cytochrome-b γ-subunit of W. succinogenes hydrogenase [8], along with the large and small subunits. It is less clear whether any of the other hyb gene products are also part of the active enzyme, indeed the hybD gene product appears to be a protease required for releasing a C-terminal peptide from the large subunit following nickel acquisition [10]. HybA appears to have no homologues in the gene clusters for the well characterised A. eutrophus or Bradyrhizobium japonicum hydrogenases [37,43].…”

“…Hydrogenase 3 is encoded by an operon (hyc) located at 58 min on the E. coli genome, which is composed of nine open reading frames hycABCDEF-GHI [4,10]. The HycE gene encodes the nickel-containing large subunit.…”

hyp Gene Products in Alcaligenes Eutrophus are part of a Hydrogenase‐Maturation System

“…However, C-terminal proteolytic processing was shown to be essential for acquisition of enzymatic activity of the Azotobucter vinelundii hydrogenase (Collin et al, 1992), the E. coli hydrogenase 3 (Rossmann et al, 1994) and the selenium-containing hydrogenase of the archaeon Methunococcus voltae (Sorggenfrei et al, 1993). The specific proteolysis is mediated by an unusual protease which has recently been purified and characterised from E. coli (Rossmann et al, 1995 the hydrogenase 3 operon. Homologous genes were identified in a number of organisms including A. eutrophus (Kortliike et al, 1992).…”

Properties of the [NiFe]‐hydrogenase maturation protein HypD

“…The synthesis and insertion of the NiFe(CO)(CN) 2 metal centre depends on the activities of a series of proteins. The core machinery, as demonstrated for the synthesis of the intracellularly located hydrogenase 3 from E. coli, consists of the products of the six hyp genes plus an endopeptidase ( [1][2][3][4] and for review see [5,6]). The two hyp gene products HypA and HypB together with the auxiliary protein SlyD [7][8][9] are required for nickel sequestration and incorporation, whereas the four other Hyp proteins (HypC to HypF) are involved in synthesis of the CN ligand and the cyanation of the active site iron [10][11][12][13].…”