Dataset Information

Stabilizing AqdC, a Pseudomonas Quinolone Signal-Cleaving Dioxygenase from Mycobacteria, by FRESCO-Based Protein Engineering.

ABSTRACT: The mycobacterial PQS dioxygenase AqdC, a cofactor-less protein with an ?/?-hydrolase fold, inactivates the virulence-associated quorum-sensing signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) produced by the opportunistic pathogen Pseudomonas aeruginosa and is therefore a potential anti-virulence tool. We have used computational library design to predict stabilizing amino acid replacements in AqdC. While 57 out of 91 tested single substitutions throughout the protein led to stabilization, as judged by increases in Tappm of >2?°C, they all impaired catalytic activity. Combining substitutions, the proteins AqdC-G40K-A134L-G220D-Y238W and AqdC-G40K-G220D-Y238W showed extended half-lives and the best trade-off between stability and activity, with increases in Tappm of 11.8 and 6.1?°C and relative activities of 22 and 72?%, respectively, compared to AqdC. Molecular dynamics simulations and principal component analysis suggested that stabilized proteins are less flexible than AqdC, and the loss of catalytic activity likely correlates with an inability to effectively open the entrance to the active site.

SUBMITTER: Wullich SC

PROVIDER: S-EPMC7894191 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Publications

Stabilizing AqdC, a Pseudomonas Quinolone Signal-Cleaving Dioxygenase from Mycobacteria, by FRESCO-Based Protein Engineering.

Wullich Sandra C SC Wijma Hein J HJ Janssen Dick B DB Fetzner Susanne S

Chembiochem : a European journal of chemical biology 20201116 4

The mycobacterial PQS dioxygenase AqdC, a cofactor-less protein with an α/β-hydrolase fold, inactivates the virulence-associated quorum-sensing signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) produced by the opportunistic pathogen Pseudomonas aeruginosa and is therefore a potential anti-virulence tool. We have used computational library design to predict stabilizing amino acid replacements in AqdC. While 57 out of 91 tested single substitutions throughout the protein led to stabilizatio ...[more]

PMID: 33058333

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Project description:The quinolone ring is a common core structure of natural products exhibiting antimicrobial, cytotoxic, and signaling activities. A prominent example is the Pseudomonas quinolone signal (PQS), a quorum-sensing signal molecule involved in the regulation of virulence of Pseudomonas aeruginosa The key reaction to quinolone inactivation and biodegradation is the cleavage of the 3-hydroxy-4(1H)-quinolone ring, catalyzed by dioxygenases (HQDs), which are members of the α/β-hydrolase fold superfamily. The α/β-hydrolase fold core domain consists of a β-sheet surrounded by α-helices, with an active site usually containing a catalytic triad comprising a nucleophilic residue, an acidic residue, and a histidine. The nucleophile is located at the tip of a sharp turn, called the "nucleophilic elbow." In this work, we developed a search workflow for the identification of HQD proteins from databases. Search and validation criteria include an [H-x(2)-W] motif at the nucleophilic elbow, an [HFP-x(4)-P] motif comprising the catalytic histidine, the presence of a helical cap domain, the positioning of the triad's acidic residue at the end of β-strand 6, and a set of conserved hydrophobic residues contributing to the substrate cavity. The 161 candidate proteins identified from the UniProtKB database originate from environmental and plant-associated microorganisms from all domains of life. Verification and characterization of HQD activity of 9 new candidate proteins confirmed the reliability of the search strategy and suggested residues correlating with distinct substrate preferences. Among the new HQDs, PQS dioxygenases from Nocardia farcinica, N. cyriacigeorgica, and Streptomyces bingchenggensis likely are part of a catabolic pathway for alkylquinolone utilization.IMPORTANCE Functional annotation of protein sequences is a major requirement for the investigation of metabolic pathways and the identification of sought-after biocatalysts. To identify heterocyclic ring-cleaving dioxygenases within the huge superfamily of α/β-hydrolase fold proteins, we defined search and validation criteria for the primarily motif-based identification of 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases (HQD). HQDs are key enzymes for the inactivation of metabolites, which can have signaling, antimicrobial, or cytotoxic functions. The HQD candidates detected in this study occur particularly in environmental and plant-associated microorganisms. Because HQDs active toward the Pseudomonas quinolone signal (PQS) likely contribute to interactions within microbial communities and modulate the virulence of Pseudomonas aeruginosa, we analyzed the catalytic properties of a PQS-cleaving subset of HQDs and specified characteristics to identify PQS-cleaving dioxygenases within the HQD family.

Dataset Information

Stabilizing AqdC, a Pseudomonas Quinolone Signal-Cleaving Dioxygenase from Mycobacteria, by FRESCO-Based Protein Engineering.

Publications

Stabilizing AqdC, a Pseudomonas Quinolone Signal-Cleaving Dioxygenase from Mycobacteria, by FRESCO-Based Protein Engineering.

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