Biochemical and structural studies of multistep posttranslational modification system for a quinone cofactor- containing enzyme subunit

概要

Quinoheme protein amine dehydrogenase (QHNDH) is a heterotrimeric enzyme that catalyzes the oxidative deamination of primary amines in a variety of Gram-negative bacteria. The smallest 9-kDa subunit QhpC, which forms the active center, contains two unique post-translational modification structures: the quinone cofactor cysteine tryptophylquinone (CTQ) and an intramolecular thioether bond. Previous studies have shown that a radical SAM enzyme QhpD first forms three intramolecular thioether bridges between Asp/Glu and Cys residues of QhpC in the cytoplasm. Next, it is postulated that the FAD-dependent monooxygenase QhpG is involved in modification of the precursor Trp residue as an initial reaction for CTQ formation. Subtilisin-like serine peptidase QhpE then remove the leader peptide of the N-terminal 28 residues of QhpC. For QhpE and QhpG, their characteristics have not been elucidated, and three- dimensional structures have been also unknown. This study focused on the roles of QhpG and QhpE in the post-translational modification of QhpC to clarify the detailed reaction mechanisms of the individual enzymes.

First, to identify the catalytic reaction of QhpG, I expressed and purified all related proteins QhpC, QhpD, QhpE, and QhpG from Ps putida using the E. coli expression system. In vitro reaction system was constructed, and the product QhpC was analyzed by mass spectrometry. The obtained results demonstrated that QhpG catalyzed the single-turnover dihydroxylation of the CTQ-precursor, Trp43, in the protein substrate QhpC containing triple intra-peptidyl crosslinks that were pre-formed by QhpD. Interestingly, it is found that QhpCDG ternary complex formation is essential for the both reactions of QhpD and QhpG. Crystal structure of this peptidyl tryptophan dihydroxylase QhpG revealed a large pocket that can dock the crosslinked QhpC with the bound FAD situated close to the precursor tryptophan. Based on the enzyme-protein substrate docking model, the peptidyl tryptophan dihydroxylation is predicted to be catalyzed by repetition of mono hydroxylation reaction that is done by the flavoprotein monooxygenase.

As for the serine proteinase QhpE from Ps. putida, kinetic analysis of removal of the leader peptide from QhpC showed that the QhpE reacts the structure of QhpC by about 50-fold faster than the linear QhpC without crosslinks. Probably the conformational change of QhpC due to crosslink formation affects the activity of QhpE. As shown above, the QhpC still keeps the QhpC/QhpD/QhpG ternary complex even after modification reactions by QhpD and QhpG. The results exhibited that QhpE can efficiently remove the leader peptide of the crosslinked QhpC from the ternary complex, not for the linear QhpC. In addtion, the X-ray crystal structure of QhpE was determined at 1.80 Å resolution. In the active center of QhpE, there was a large pocket where the QhpC is thought to be bound. Probably the compact structure of crosslinked QhpC is recognized by this pocket. The high reactivity of QhpE toward the crosslinked QhpC is predicted to prevent the cleavage of the newly translated and uncrosslinked QhpC, facilitating the progress of QhpC biosynthesis through complex formation.

Thus, I demonstrated that multiple enzyme system regulates the biosynthesis of mature QhpC through complex formation and reactivity of each enzyme. These efficient and rational mechanisms seem essential for rapid production of the active QHNDH to assimilate primary amines under starvation conditions without carbon sources. It is expected that the posttranslational mechanism of QhpC including crosslinking and processing of peptides is applicable for developing bioactive cyclic peptides.

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