Title: Structural basis for recognition and ring-cleavage of the Pseudomonas quinolone signal (PQS) by AqdC, a mycobacterial dioxygenase of the alpha/beta-hydrolase fold family Wullich SC, Kobus S, Wienhold M, Hennecke U, Smits SHJ, Fetzner S Ref: J Struct Biol, 207:287, 2019 : PubMed
The cofactor-less dioxygenase AqdC of Mycobacteroides abscessus catalyzes the cleavage and thus inactivation of the Pseudomonas quinolone signal (PQS, 2-heptyl-3-hydroxy-4(1H)-quinolone), which plays a central role in the regulation of virulence factor production by Pseudomonas aeruginosa. We present here the crystal structures of AqdC in its native state and in complex with the PQS cleavage product N-octanoylanthranilic acid, and of mutant AqdC proteins in complex with PQS. AqdC possesses an alpha/beta-hydrolase fold core domain with additional helices forming a cap domain. The protein is traversed by a bipartite tunnel, with a funnel-like entry section leading to an elliptical substrate cavity where PQS positioning is mediated by a combination of hydrophobic interactions and hydrogen bonds, with the substrate's C4 carbonyl and C3 hydroxyl groups tethered by His97 and the catalytic His246, respectively. The side chain of the AqdC-bound product extends deeper into the "alkyl tail section" of the tunnel than PQS, tentatively suggesting product exit via this part of the tunnel. AqdC prefers PQS over congeners with shorter alkyl substituents at C2. Kinetic data confirmed the strict requirement of the active-site base His246 for catalysis, and suggested that evolution of the canonical nucleophile/His/Asp catalytic triad of the hydrolases to an Ala/His/Asp triad is favorable for catalyzing dioxygenolytic PQS ring cleavage.
Pseudomonas aeruginosa employs 2-heptyl-3-hydroxy-4(1H)-quinolone (the Pseudomonas quinolone signal, PQS) and 2-heptyl-4(1H)-quinolone (HHQ) as quorum sensing signal molecules, which contribute to a sophisticated regulatory network controlling the production of virulence factors and antimicrobials. We demonstrate that Mycobacterium abscessus(T) and clinical M. abscessus isolates are capable of degrading these alkylquinolone signals. Genome sequences of 50 clinical M. abscessus isolates indicated the presence of aqdRABC genes, contributing to fast degradation of HHQ and PQS, in M. abscessus subsp. abscessus strains, but not in M. abscessus subsp. bolletii and M. abscessus subsp. massiliense isolates. A subset of 18 M. a. subsp. abscessus isolates contained the same five single nucleotide polymorphisms (SNPs) compared to the aqd region of the type strain. Interestingly, representatives of these isolates showed faster PQS degradation kinetics than the M. abscessus type strain. One of the SNPs is located in the predicted promoter region of the aqdR gene encoding a putative transcriptional regulator, and two others lead to a variant of the AqdC protein termed AqdC(II), which differs in two amino acids from AqdC(I) of the type strain. AqdC, the key enzyme of the degradation pathway, is a PQS dioxygenase catalyzing quinolone ring cleavage. While transcription of aqdR and aqdC is induced by PQS, transcript levels in a representative of the subset of 18 isolates were not significantly altered despite the detected SNP in the promoter region. However, purified recombinant AqdC(II) and AqdC(I) exhibit different kinetic properties, with approximate apparent Km values for PQS of 14 muM and 37 muM, and kcat values of 61 s(-1) and 98 s(-1), respectively, which may (at least in part) account for the observed differences in PQS degradation rates of the strains. In co-culture experiments of P. aeruginosa PAO1 and M. abscessus, strains harboring the aqd genes reduced the PQS levels, whereas mycobacteria lacking the aqd gene cluster even boosted PQS production. The results suggest that the presence and expression of the aqd genes in M. abscessus lead to a competitive advantage against P. aeruginosa.