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

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 smallest ␥-subunit of the ␣␤␥ heterotrimeric protein. A putative [Fe-S]-cluster-binding protein (ORF2 protein) encoded between the structural genes for the ␣-and ␥-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 ␣-and ␤-subunits were both detected in the periplasm of the mutant strain, whereas the ␥-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 ␥-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 ␥-subunit, most likely participating in the formation of the intrapeptidyl thioether cross-links. 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 (1-3). QHNDH contains a posttranslationally derived quinone cofactor, cysteine tryptophylquinone (CTQ) (Fig. 1A), within the smallest ␥-subunit of the ␣␤␥ heterotrimeric protein (4 -6). The largest ␣-subunit folds into four domains containing two c-type hemes in the first domain (5, 6). The ␤-subunit has a seven-bladed ␤-propeller structure often observed in quinoproteins. CTQ is the most recently identified, fourth quinone cofactor derived from an amino acid residue(s) constituting the polypeptide chain after the first identified topa quinone of copper amine oxidase (7), the second identified tryptophan tryptophylquinone (TTQ) (Fig. 1B) of methylamine dehydrogenase (MADH) (8), and the third identified lysine tyrosylquinone of lysyl oxidase (9). In addition to CTQ that is essential as a redox cofactor for the catalytic reaction (1...

The Radical S-Adenosyl-l-methionine Enzyme QhpD Catalyzes Sequential Formation of Intra-protein Sulfur-to-Methylene Carbon Thioether Bonds

“…QhpD is essential for the biogenesis of QHNDH, most likely by participating in the Cys-to-Asp/Glu thioether bond formation in the ␥ subunit (maturated QhpC) (7). Consistent with its indispensable role in the QHNDH biogenesis, the qhpD gene is strictly conserved in all bacteria possessing the qhp operon with the common gene array, qhpADCB (and their reverse, BCDA, in the complementary strands) (4).…”

“…However, QhpD differs from these proteins in that it acts on an enzyme subunit to form a multiknotted structure of the polypeptide chain with sulfur-to-methylene carbon thioether crosslinks (7). In this study, we describe efficient expression of recombinant QhpD from Paracoccus denitrificans in complex with its substrate QhpC and biochemical characterization as a radical SAM enzyme, and we demonstrate that QhpD does catalyze the formation of intra-protein sulfur-to-methylene carbon thioether bonds in QhpC.…”

“…Three other thioether cross-links (Cys-7-Glu-16, Cys-27-Asp-33, and Cys-41-Asp-49) are formed between the sulfur atom of a * This work was supported by Japan Society for the Promotion of Science Cys residue and the methylene carbon atom of an Asp or Glu residue, affording the structural stabilization of the subunit polypeptide chain that is otherwise a featureless coil with only two short ␣-helices. Therefore, the ␥ subunit must undergo multiple post-translational modifications, in which many gene products encoded in the qhp operon, including QhpD, QhpE, QhpF, and QhpG, are involved (4,7,8), and such events occur before or after associating with the ␣ and ␤ subunits to form the active QHNDH complex.…”

The Silent Form of Quinohemoprotein Amine Dehydrogenase fromParacoccus denitrificans

“…[12][13][14] According to CTQ, putative iron sulfur cluster binding protein probably belongs to the Radical SAM superfamily and can be involved in the biogenesis of QHAmDH, 11) but the details remain obscure. If sQHAmDH is the precursor of QH-AmDH, sQH-AmDH might become a key protein in understanding the biogenesis of post-transcriptionally derived quinone cofactors.…”

“…9,10) The biosynthesis of CTQ requires at least one extra gene, and it is different from those required for TTQ biosynthesis. 11) In TTQ biosynthesis, MauG protein, an oxygen-binding diheme c protein, would be involved in cross-linking reaction of the two tryptophans. [12][13][14] The largest subunit of QH-AmDH is a 489-residue, a four-domain polypeptide chain that contains two heme c cofactors.…”