Involvement of a Putative [Fe-S]-cluster-binding Protein in the Biogenesis of Quinohemoprotein Amine Dehydrogenase

Kazutoshi Ono,Toshihide Okajima,Minobu Tani,Shun'Ichi Kuroda,Dapeng Sun,Victor L Davidson,Katsuyuki Tanizawa

doi:10.1074/jbc.m600029200

Kazutoshi Ono, Toshihide Okajima + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m600029200

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Abstract

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.

Highlights

Quinohemoprotein amine dehydrogenase (QHNDH)4 is produced in the periplasmic space of certain Gram-negative bacteria, such as Paracoccus denitrificans and Pseudomonas putida, in response to primary amines, including n-butylamine and benzylamine, added to the culture medium as a sole carbon and energy source and catalyzes their oxidative deamination [1,2,3]
Effect of Disruption of ORF2 Gene on Bacterial Growth and QHNDH Activity—When the wild-type P. denitrificans Pd1222 strain was cultured in the medium containing n-butylamine as a sole carbon and energy source, the bacterium grew efficiently by inducibly forming QHNDH (Fig. 2A), which catalyzes the 2eϪ oxidation of n-butylamine, as reported previously for the P. denitrificans IFO12442 strain [1]
These results clearly show that the ORF2 protein is not hypothetical but expressed as a protein to be involved in the biogenesis of active QHNDH that facilitates the bacterial cell growth in the n-butylamine medium

Summary

EXPERIMENTAL PROCEDURES

Materials—Restriction endonucleases, DNA modifying enzymes, and endoproteinase Asp-N were purchased from Takara Bio. To construct an expression plasmid for only the ORF2 protein, the ORF2 gene was amplified by PCR using the above-constructed pGEM-T containing the entire QHNDH gene as a template with primers P11 (5Ј-ATCCATATGCGACGGTGCAGCGCTTC-3Ј and P12 (5Ј-CCGGGATCCTCATTGGAGAACCTCCCTTGC-3Ј) in which NdeI and BamHI sites (underlined) were introduced, respectively. Isolation of ␥-Subunit Polypeptide—P. denitrificans PdKO4 was cultured in the medium containing n-butylamine and choline, and the spheroplasts prepared as described above were washed twice with phosphate-buffered saline and resuspended at a final concentration of 0.1 g/ml in 20 mM Tris-HCl buffer, pH 8.0, containing 0.1 M dithiothreitol and 10 mM EDTA1⁄72Na. The spheroplasts were disrupted by sonication, and the supernatant was obtained by centrifugation at 50,000 ϫ g for 60 min. Tide peaks in MALDI-TOF mass spectra were assigned using a FindPept tool

RESULTS

2–82 Ϫ28–82

12–32 Ϫ28–11 Ϫ28–11 Ϫ28–11 Ϫ28–11

DISCUSSION