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  • An unusual subtilisin-like serine protease is essential for biogenesis of quinohemoprotein amine dehydrogenase.

An unusual subtilisin-like serine protease is essential for biogenesis of quinohemoprotein amine dehydrogenase.

The Journal of biological chemistry (2012-01-12)
Tadashi Nakai, Kazutoshi Ono, Shun'ichi Kuroda, Katsuyuki Tanizawa, Toshihide Okajima
摘要

Quinohemoprotein amine dehydrogenase (QHNDH), an αβγ heterotrimer present in the periplasm of several Gram-negative bacteria, catalyzes the oxidative deamination of various aliphatic amines such as n-butylamine for assimilation as carbon and energy sources. The γ subunit of mature QHNDH contains a protein-derived quinone cofactor, cysteine tryptophylquinone, and three intrapeptidyl thioether cross-links between Cys and Asp or Glu residues. In its cytoplasmic nascent form, the γ subunit has a 28-residue N-terminal leader peptide that is necessary for the production of active QHNDH but must be removed in the following maturation process. Here, we describe the role of a subtilisin-like serine protease encoded in the fifth ORF of the n-butylamine-utilizing operon of Paracoccus denitrificans (termed ORF5) in QHNDH biogenesis. ORF5 disruption caused bacterial cell growth inhibition in n-butylamine-containing medium and production of inactive QHNDH, in which the γ subunit retained the leader peptide. Supply of plasmid-encoded ORF5 restored the cell growth and production of active QHNDH, containing the correctly processed γ subunit. ORF5 expressed in Escherichia coli but not its catalytic triad mutant cleaved synthetic peptides surrogating for the γ subunit leader peptide, although extremely slowly. The cleaved leader peptide remained unstably bound to ORF5, most likely as an acyl enzyme intermediate attached to the active-site Ser residue. These results demonstrate that ORF5 is essential for QHNDH biogenesis, serving as a processing protease to cleave the γ subunit leader peptide nearly in a disposable manner.

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