Involvement of a putative [Fe-S]-cluster-binding protein in the biogenesis of quinohemoprotein amine dehydrogenase.
Quinohemoprotein amine dehydrogenase (QHNDH) of Paracoccus denitrificans contains a peptidyl quinone cofactor, cysteine tryptophylquinone, as well as intrapeptidyl thioether cross-links between Cys and Asp/Glu residues within the smallestgamma-subunit of the alphabetagamma heterotrimeric protein. A putative [Fe-S]-cluster-binding protein (ORF2 protein) encoded between the structural genes for the alpha- and gamma-subunits of QHNDH in the n-butylamine-utilizing operon likely belongs to a Radical SAM (S-Ado-Met) superfamily that includes many proteins involved in vitamin biosynthesis and enzyme activation. In this study the role of ORF2 protein in the biogenesis of QHNDH has been explored. Although the wild-type strain of Paracoccus denitrificans produced an active, mature enzyme upon induction with n-butylamine, a mutant strain in which the ORF2 gene had been mostly deleted, neither grew in the n-butylamine medium nor showed QHNDH activity. When the mutant strain was transformed with an expression plasmid for the ORF2 protein, n-butylamine-dependent bacterial growth and QHNDH activity were restored. Site-specific mutations in the putative [Fe-S]-cluster or SAM binding motifs in the ORF2 protein failed to support bacterial growth. The alpha- and beta-subunits were both detected in the periplasm of the mutant strain, whereas the gamma-subunit polypeptide was accumulated in the cytoplasm and stained negatively for redox-cycling quinone staining. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis revealed that the gamma-subunit isolated from the mutant strain had not undergone posttranslational modification. These results unequivocally show that the putative [Fe-S]-cluster- and SAM-binding ORF2 protein is necessary for the posttranslational processing of gamma-subunit, most likely participating in the formation of the intrapeptidyl thioether cross-links.
Ono
Kazutoshi
K
Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan.
Okajima
Toshihide
T
Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan. Electronic address: tokajima@sanken.osaka-u.ac.jp.
Tani
Minobu
M
Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan.
Kuroda
Shun'ichi
S
Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan.
Sun
Dapeng
D
Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi 39216-4505.
Davidson
Victor L
VL
Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi 39216-4505.
Tanizawa
Katsuyuki
K
Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan. Electronic address: tanizawa@sanken.osaka-u.ac.jp.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000096:biosynthesis*; D010231:Paracoccus denitrificans / Q000201:enzymology; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D021122:Protein Subunits; D000587:Oxidoreductases Acting on CH-NH Group Donors; C119497:quinohemoprotein amine dehydrogenase