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J. Bacteriol., 04 1996, 2368-2374, Vol 178, No. 8
Copyright © 1996, American Society for Microbiology

Carboxyl-terminal processing of the cytoplasmic NAD-reducing hydrogenase of Alcaligenes eutrophus requires the hoxW gene product

S Thiemermann, J Dernedde, M Bernhard, W Schroeder, C Massanz and B Friedrich
Institut fur Pflanzenphysiologie und Mikrobiologie, Freie Universitat Berlin, Germany.

Two open reading frames (ORFs) were identified immediately downstream of the four structural genes for the soluble hydrogenase (SH) of Alcaligenes eutrophus H16. While a mutation in ORF2 had no obvious effect on hydrogen metabolism, an in-frame deletion in ORF1, subsequently designated hoxW, led to a complete loss of SH activity and hence a significant retardation of autotrophic growth on hydrogen. Hydrogen oxidation in the hoxW mutant was catalyzed by the second hydrogenase, a membrane-bound enzyme. Assembly of the four subunits of the SH was blocked in mutant cells, and HoxH, the hydrogen-activating subunit, accumulated as a precursor which was still capable of binding nickel. Protein sequencing revealed that HoxH isolated from the wild type terminates at His-464, whereas the C-terminal amino acid sequence of HoxH from the hoxW mutant is colinear with the deduced sequence. Processing of the HoxH precursor was restored in vitro by a cell extract containing HoxW. These results indicate that HoxW is a highly specific carboxyl-terminal protease which releases a 24-amino-acid peptide from HoxH prior to progression of subunit assembly.

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