title: An unusual subtilisin-like serine protease is essential for biogenesis of quinohemoprotein amine dehydrogenase.
abstract: 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.
authors.affiliation: Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Nakai
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Ono
authors.forename: Shun'ichi
authors.initials: S
authors.lastname: Kuroda
authors.forename: Katsuyuki
authors.initials: K
authors.lastname: Tanizawa
authors.forename: Toshihide
authors.initials: T
authors.lastname: Okajima
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000502:physiology; D016366:Open Reading Frames / Q000235:genetics; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000235:genetics*; D010231:Paracoccus denitrificans / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D010957:Plasmids / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000502:physiology; D017434:Protein Structure, Tertiary; D057057:Serine Proteases / Q000235:genetics* / Q000378:metabolism*; D020860:Subtilisin / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D000587:Oxidoreductases Acting on CH-NH Group Donors; C119497:quinohemoprotein amine dehydrogenase; D057057:Serine Proteases; D020860:Subtilisin