C-terminal extension of the H2-activating subunit, HoxH, directs maturation of the NAD-reducing hydrogenase in Alcaligenes eutrophus.
Formation of enzymatically active [NiFe] hydrogenases is dependent on a number of posttranslational steps, including metal attachment to a precursor of the catalytic subunit, truncation of a small C-terminal peptide from the precursor, and oligomerisation of the subunits. Two amino acid replacements were introduced by site-directed mutagenesis at the C-terminal proteolytic cleavage site of HoxH, the Ni-containing subunit of the cytoplasmic NAD-reducing hydrogenase of Alcaligenes eutrophus H16. Replacement of Ala465, the first residue of the 24-amino-acid cleaved polypeptide, by Pro yielded a form of HoxH that was blocked in C-terminal proteolysis. This HoxH subunit, although capable of binding Ni, was blocked in formation of a stable tetrameric holoenzyme. In the second mutant, the C-terminal extension of HoxH was eliminated by substituting the Ala codon for a translational stop codon. Although this mutant subunit was able to form the oligomeric holoenzyme, it was devoid of Ni. Both mutant proteins contained only traces of H2-activating functions. H2-dependent reduction of NAD and benzylviologen, and D2/H+-exchange activity were almost completely abolished, while the NADH oxidoreductase activity, mediated by the diaphorase moiety of the hydrogenase, was retained. These results allow the following conclusions: the C-terminal extension of HoxH is neccessary to direct specific Ni insertion into the hydrogenase; subunit assembly to the holoenzyme is not dependent on Ni insertion; and a precursor with the C-terminal peptide is not competent for assembly.
Massanz
C
C
Institut für Biologie der Humboldt-Universitat zu Berlin, Germany.
Fernandez
V M
VM
Friedrich
B
B
D000421:Alcaligenes / Q000201:enzymology*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D004269:DNA, Bacterial / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006859:Hydrogen / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009532:Nickel / Q000378:metabolism; D009876:Operon; D010088:Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D015183:Restriction Mapping; D012995:Solubility
D000596:Amino Acids; D004269:DNA, Bacterial; D009532:Nickel; D006859:Hydrogen; D010088:Oxidoreductases; C021376:hydrogen dehydrogenase