Maturation of the large subunit (HYCE) of Escherichia coli hydrogenase 3 requires nickel incorporation followed by C‐terminal processing at Arg537

Abstract

Purification of the large subunit, HYCE, of Escherichia coli hydrogenase 3 revealed that it is a nickel-containing polypeptide, which is subject to C-terminal proteolytic processing. This processing reaction could be performed in vitro with partially purified components, yielding a low-molecular mass C-terminal peptide which was resolved in a Tricine/SDS/polyacrylamide gel. N-terminal sequencing of this peptide revealed that proteolytic cleavage occurred at the C-terminal side of the arginine residue at position 537, which corresponds to the histidine residue in the highly conserved motif, DPCXXCXXH, of other (NiFe) hydrogenases thought to be involved in active site nickel coordination. Nickel-containing HYCE precursor for in vitro processing, was partially purified from strain HD708 (AhycH) in the presence of the reducing agent dithiothreitol. Using 2-mercaptoethanol instead of dithiothreitol provided pure precursor, which was, however, no longer susceptible to in vitro processing ; it proved to be devoid of nickel indicating that nickel incorporation into the HYCE precursor is a prerequisite for processing. This conclusion was supported by the finding that HYCE precursor from strain HD708 (AhycH) chromatographed with radioactivity from 63Ni incorporated in vivo and could be processed in vitro, whereas HYCE precursor from strain BEF314 (AhypB-E) lacking the nickel insertion system appeared to be devoid of nickel and was not sensitive to in vitro processing.According to their different physiological roles in bacterial metabolism, nickel-containing hydrogenases form a very heterogeneous group of enzymes. They differ with respect to their cellular function (H, oxidation or H, production), metal content, subcellular localization, subunit composition and the electron acceptors and donors used [l]. However, one subunit, called the large subunit, is common to all of these hydrogenases. In the amino acid sequence, it is the most conserved subunit and it is thought to ligand the active-site nickel. Two highly conserved cysteine-rich motifs are present in all known large subunits and probably provide the ligands required for the binding of nickel. One motif, RXCXXC, is located in the N-terminal region and the second, DPCXXCXXH, is located at the C-terminus of the polypeptide chain [l, 21. In addition to the genes encoding the hydrogenase subunits, a number of genes have been sequenced which are required for the formation of active hydrogenases [ 3 -101. Such activation of (NiFe) hydrogenases may involve insertion of nickel into the hydrogenase apoprotein [ l l -131, activation of nickel in the active site, membrane integration [3, 141, complex formation, processing of the small subunit carrying a signal peptide at its N-terminus [15] and processing of the large subunit [3,16, 171 which order these processes take place and whether further reactions are involved.The facultative anaerobe Escherichia coli possesses three hydrogenase isoenzymes [ 181. Two of these isoenzymes (hydrogenases 1 and 2) are membrane-bound uptake hy...

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

“…Such leader peptides have not been detected in the large subunits. 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.…”

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

“…E-mail: wu@ibsm.cnrs-mrs.fr HYD2 and HYD3 activities, respectively [2][3][4][5]. All three hydrogenases contain nickel [1,[6][7][8].…”

“…The processing of the large subunits of the hydrogenase from Azotobacter vinelandii [10], of HYD3 from E. coil [8], and of the periplasmic hydrogenase from Desulfovibrio gigas [11] results from a proteolytic cleavage after a histidine or an arginine residue in the conserved motif Asp-Pro-CysXaa-Xaa-Cys-Xaa-Xaa-His/Arg (DPCxxCxxH/R) located at the C-termini of the large subunits. The highly conserved histidyl residue is naturally substituted by an arginine in the HYD3 of E. coli [8]. 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 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]. The processing of the large subunit of HYD3 is correlated with the nickel incorporation into the polypeptide chain [8]. The Cterminal extension in the precursor form of the large subunit of HYD3 has recently been shown to keep the protein in a conformation required for the liganding of the metal [13].…”

Solution Structure and Backbone Dynamics of an Endopeptidase HycI from scherichia coli

“…In addition, there are three 3 10 -helices (residues Asp-15-Gly-17 (3 10 -1), Pro-43-Asp-46 (3 10 -2), and Met-86-Thr-88 (3 10 -3) distributed on the surface and proximate to the putative nickelbinding site. Fig.…”