An Unusual Subtilisin-like Serine Protease Is Essential for Biogenesis of Quinohemoprotein Amine Dehydrogenase

Abstract

Background: Quinohemoprotein amine dehydrogenase from Gram-negative Proteobacteria undergoes multiple posttranslational modifications. Results: Disruption of a neighboring gene encoding a subtilisin-like serine protease led to the production of inactive dehydrogenase. Conclusion:The protease is essential for biogenesis of the dehydrogenase, serving as an unusual cis-acting enzyme without catalytic turnover. Significance: The protease described here may represent a rare bacterial version of processing enzymes.

Intrigues and Intricacies of the Biosynthetic Pathways for the Enzymatic Quinocofactors: PQQ, TTQ, CTQ, TPQ, and LTQ

“…It is postulated that the latter may play a role in the export of QHNDH or its subunits from the cytoplasm to periplasm. 86 …”

“…The activity of the protease toward short peptides has shown only limited or single turnover kinetics, and this has allowed the identification of a covalent adduct between the ORF 5 protein and the N-terminal of the cleaved peptide, further confirming a function for the CTQ-linked protease that falls squarely within the category of serine proteases. 86 Based on the available data, a tentative pathway for the sequence of events in CTQ production is outlined in Scheme 6.…”

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

“…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.…”

“…3). For QhpC, we also prepared a series of full-length and truncated forms with or without the N-terminal 28-residue leader peptide that is essential for the formation of thioether bonds in QhpC (7) but must be removed subsequently by a subtilisin-like protease QhpE (8). Initial attempts to express QhpD alone or by co-expression with a plasmid carrying E. coli iscRSUA-hscBAfdx-iscX genes (30) or sufABCDSE genes (31), which are expected to assist the assembly of iron-sulfur clusters in radical SAM proteins (21), were all unsuccessful for efficient production of QhpD in the soluble fraction of the E. coli C41 (DE3) cell lysate.…”

Two Different Quinohemoprotein Amine Dehydrogenases Initiate Anaerobic Degradation of Aromatic Amines in Aromatoleum aromaticum EbN1

“…The observed N-terminal peptide is contained in the predicted signal sequence of the ␥-subunit, suggesting only partial proteolytic processing in the mature enzyme. The ␥-subunits of quinohemoproteins of this family are expected to be heavily modified by the products of several further genes in the respective operons, which insert four thioether cross-links involving the four cysteines present in the sequence (C36, C56, C65, and C69) and generate a CTQ cofactor (25,26). The internal thioether cross-links appear to impede detection via SDS-PAGE, because the peptide chains cannot be unfolded by SDS and prevent the production of small tryptic fragments from the central part of the subunits.…”