Stabilizing AqdC, a <i>Pseudomonas</i> Quinolone Signal‐Cleaving Dioxygenase from Mycobacteria, by FRESCO‐Based Protein Engineering

Wullich, Sandra C.; Wijma, Hein J.; Janssen, Dick B.; Fetzner, Susanne

doi:10.1002/cbic.202000641

Cited by 6 publications

(7 citation statements)

References 74 publications

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“…Previous studies indicated that the characteristics of substrate access tunnels can have a decisive influence on enzyme–substrate specificity and activity. − In t-PlaF, we focused on T1 and T2 because only these two allow direct access of GPL or LGPL substrates from the membrane in the t-PlaF A configuration. In contrast, to enter into T3, substrates would need to pass through the solvent, which is energetically unfavorable.…”

Section: Discussionmentioning

confidence: 99%

Substrate Access Mechanism in a Novel Membrane-Bound Phospholipase A of Pseudomonas aeruginosa Concordant with Specificity and Regioselectivity

Ahmad

Strunk

Schott‐Verdugo

et al. 2021

J. Chem. Inf. Model.

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PlaF is a cytoplasmic membrane-bound phospholipase A 1 from Pseudomonas aeruginosa that alters the membrane glycerophospholipid (GPL) composition and fosters the virulence of this human pathogen. PlaF activity is regulated by a dimer-to-monomer transition followed by tilting of the monomer in the membrane. However, how substrates reach the active site and how the characteristics of the active site tunnels determine the activity, specificity, and regioselectivity of PlaF for natural GPL substrates have remained elusive. Here, we combined unbiased and biased all-atom molecular dynamics (MD) simulations and configurational free-energy computations to identify access pathways of GPL substrates to the catalytic center of PlaF. Our results map out a distinct tunnel through which substrates access the catalytic center. PlaF variants with bulky tryptophan residues in this tunnel revealed decreased catalysis rates due to tunnel blockage. The MD simulations suggest that GPLs preferably enter the active site with the sn-1 acyl chain first, which agrees with the experimentally demonstrated PLA 1 activity of PlaF. We propose that the acyl chain-length specificity of PlaF is determined by the structural features of the access tunnel, which results in favorable free energy of binding of medium-chain GPLs. The suggested egress route conveys fatty acid (FA) products to the dimerization interface and, thus, contributes to understanding the product feedback regulation of PlaF by FA-triggered dimerization. These findings open up opportunities for developing potential PlaF inhibitors, which may act as antibiotics against P. aeruginosa.

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Section: Discussionmentioning

confidence: 99%

Substrate Access Mechanism in a Novel Membrane-Bound Phospholipase A of Pseudomonas aeruginosa Concordant with Specificity and Regioselectivity

Ahmad

Strunk

Schott‐Verdugo

et al. 2021

J. Chem. Inf. Model.

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show abstract

“…Previous studies indicated that the characteristics of substrate access tunnels can have a decisive influence on enzyme-substrate specificity and activity [53][54][55][56]. In t-PlaF, we focused on T1 and T2 because only these two allow direct access of GPL or LGPL substrates from the membrane in the t-PlaF A configuration.…”

Section: Discussionmentioning

confidence: 99%

Substrate access mechanism in a novel membrane-bound phospholipase A of Pseudomonas aeruginosa concordant with specificity and regioselectivity

Ahmad

Strunk

Schott‐Verdugo

et al. 2021

Preprint

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PlaF is a cytoplasmic membrane-bound phospholipase A1 from Pseudomonas aeruginosa that alters the membrane glycerophospholipid (GPL) composition and fosters the virulence of this human pathogen. PlaF activity is regulated by a dimer-to-monomer transition followed by tilting of the monomer in the membrane. However, how substrates reach the active site and how the characteristics of the active site tunnels determine the activity, specificity, and regioselectivity of PlaF for natural GPL substrates has remained elusive. Here, we combined unbiased and biased all-atom molecular dynamics (MD) simulations and configurational free energy computations to identify access pathways of GPL substrates to the catalytic center of PlaF. Our results map out a distinct tunnel through which substrates access the catalytic center. PlaF variants with bulky tryptophan residues in this tunnel revealed decreased catalysis rates due to tunnel blockage. The MD simulations suggest that GPLs preferably enter the active site with the sn-1 acyl chain first, which agrees with the experimentally demonstrated PLA1 activity of PlaF. We propose that the acyl chain-length specificity of PlaF is determined by the structural features of the access tunnel, which results in favorable free energy of binding of medium-chain GPLs. The suggested egress route conveys fatty acid products to the dimerization interface and, thus, contributes to understanding the product feedback regulation of PlaF by fatty acid-triggered dimerization. These findings open up opportunities for developing potential PlaF inhibitors, which may act as antibiotics against P. aeruginosa.

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“…AqdC VIII Mycobacteroides abscessus ATCC 19977 CAM60402.1 pBAD::his8-tev-aqdC-G40K-G220D-Y238W [19] HQD N.f. Nocardia farcinica IFM 10152 BAD60071.1 pET28b::his8-TEV-HQD N.f.…”

Section: Bacterial Strains Hqd Enzymes and Plasmidsmentioning

confidence: 99%

“…A main drawback of using HodC or AqdC for potential application as QQ enzymes is their susceptibility to proteolytic degradation or denaturation in adverse physicochemical conditions, respectively [ 15 , 16 ]. To improve the robustness of mycobacterial AqdC, a recent study used computational library design to predict stabilizing amino acid replacements and to engineer more thermostable variants [ 19 ].…”

Section: Introductionmentioning

confidence: 99%

Enzyme-Mediated Quenching of the Pseudomonas Quinolone Signal (PQS): A Comparison between Naturally Occurring and Engineered PQS-Cleaving Dioxygenases

et al. 2022

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The opportunistic pathogen Pseudomonas aeruginosa employs quorum sensing to govern the production of many virulence factors. Interference with quorum sensing signaling has therefore been put forward as an attractive approach to disarm this pathogen. Here, we analyzed the quorum quenching properties of natural and engineered (2-alkyl-)3-hydroxy-4(1H)-quinolone 2,4-dioxygenases (HQDs) that inactivate the P. aeruginosa signal molecule PQS (Pseudomonas quinolone signal; 2-heptyl-3-hydroxy-4(1H)-quinolone). When added exogenously to P. aeruginosa cultures, all HQDs tested significantly reduced the levels of PQS and other alkylquinolone-type secondary metabolites deriving from the biosynthetic pathway, such as the respiratory inhibitor 2-heptyl-4-hydroxyquinoline N-oxide. HQDs from Nocardia farcinica and Streptomyces bingchenggensis, which combine low KM values for PQS with thermal stability and resilience in the presence of P. aeruginosa exoproducts, respectively, attenuated production of the virulence factors pyocyanin and pyoverdine. A delay in mortality was observed when Galleria mellonella larvae were infected with P. aeruginosa suspensions treated with the S. bingchenggensis HQD or with inhibitors of alkylquinolone biosynthesis. Our data indicate that quenching of PQS signaling has potential as an anti-virulence strategy; however, an efficient anti-virulence therapy against P. aeruginosa likely requires a combination of agents addressing multiple targets.

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

Cited by 6 publications

References 74 publications

Substrate Access Mechanism in a Novel Membrane-Bound Phospholipase A of Pseudomonas aeruginosa Concordant with Specificity and Regioselectivity

Substrate Access Mechanism in a Novel Membrane-Bound Phospholipase A of Pseudomonas aeruginosa Concordant with Specificity and Regioselectivity

Substrate access mechanism in a novel membrane-bound phospholipase A of Pseudomonas aeruginosa concordant with specificity and regioselectivity

Enzyme-Mediated Quenching of the Pseudomonas Quinolone Signal (PQS): A Comparison between Naturally Occurring and Engineered PQS-Cleaving Dioxygenases

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