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

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 stabilization, as judged by increases in (Formula presented.) 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 (Formula presented.) 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

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

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 K(M) 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

Nickel quercetinase, a “promiscuous” metalloenzyme: metal incorporation and metal ligand substitution studies

Background: Quercetinases are metal-dependent dioxygenases of the cupin superfamily. While fungal quercetinases are copper proteins, recombinant Streptomyces quercetinase (QueD) was previously described to be capable of incorporating Ni2+ and some other divalent metal ions. This raises the questions of which factors determine metal selection, and which metal ion is physiologically relevant. Results: Metal occupancies of heterologously produced QueD proteins followed the order Ni > Co > Fe > Mn. Iron, in contrast to the other metals, does not support catalytic activity. QueD isolated from the wild-type Streptomyces sp. strain FLA contained mainly nickel and zinc. In vitro synthesis of QueD in a cell-free transcription-translation system yielded catalytically active protein when Ni2+ was present, and comparison of the circular dichroism spectra of in vitro produced proteins suggested that Ni2+ ions support correct folding. Replacement of individual amino acids of the 3His/1Glu metal binding motif by alanine drastically reduced or abolished quercetinase activity and affected its structural integrity. Only substitution of the glutamate ligand (E76) by histidine resulted in Ni- and Co-QueD variants that retained the native fold and showed residual catalytic activity. Conclusions: Heterologous formation of catalytically active, native QueD holoenzyme requires Ni2+, Co2+ or Mn2+, i.e., metal ions that prefer an octahedral coordination geometry, and an intact 3His/1Glu motif or a 4His environment of the metal. The observed metal occupancies suggest that metal incorporation into QueD is governed by the relative stability of the resulting metal complexes, rather than by metal abundance. Ni2+ most likely is the physiologically relevant cofactor of QueD of Streptomyces sp. FLA.<br

Complete genome sequence and metabolic potential of the quinaldine-degrading bacterium Arthrobacter sp. Rue61a

Background: Bacteria of the genus Arthrobacter are ubiquitous in soil environments and can be considered as true survivalists. Arthrobacter sp. strain Rue61a is an isolate from sewage sludge able to utilize quinaldine (2- methylquinoline) as sole carbon and energy source. The genome provides insight into the molecular basis of the versatility and robustness of this environmental Arthrobacter strain. Results: The genome of Arthrobacter sp. Rue61a consists of a single circular chromosome of 4,736,495 bp with an average G + C content of 62.32%, the circular 231,551-bp plasmid pARUE232, and the linear 112,992-bp plasmid pARUE113 that was already published. Plasmid pARUE232 is proposed to contribute to the resistance of Arthrobacter sp. Rue61a to arsenate and Pb2+, whereas the linear plasmid confers the ability to convert quinaldine to anthranilate. Remarkably, degradation of anthranilate exclusively proceeds via a CoA-thioester pathway. Apart from quinaldine utilization, strain Rue61a has a limited set of aromatic degradation pathways, enabling the utilization of 4-hydroxy-substituted aromatic carboxylic acids, which are characteristic products of lignin depolymerization, via ortho cleavage of protocatechuate. However, 4-hydroxyphenylacetate degradation likely proceeds via meta cleavage of homoprotocatechuate. The genome of strain Rue61a contains numerous genes associated with osmoprotection, and a high number of genes coding for transporters. It encodes a broad spectrum of enzymes for the uptake and utilization of various sugars and organic nitrogen compounds. A. aurescens TC-1 is the closest sequenced relative of strain Rue61a. Conclusions: The genome of Arthrobacter sp. Rue61a reflects the saprophytic lifestyle and nutritional versatility of the organism and a strong adaptive potential to environmental stress. The circular plasmid pARUE232 and the linear plasmid pARUE113 contribute to heavy metal resistance and to the ability to degrade quinaldine, respectively

Biotic inactivation of the Pseudomonas aeruginosa quinolone signal molecule

In Pseudomonas aeruginosa, quorum sensing (QS) regulates the production of secondary metabolites, many of which are antimicrobials that impact on polymicrobial community composition. Consequently, quenching QS modulates the environmental impact of P. aeruginosa. To identify bacteria capable of inactivating the QS signal molecule 2-heptyl-3- hydroxy-4(1H)-quinolone (PQS), a minimal medium containing PQS as the sole carbon source was used to enrich a Malaysian rainforest soil sample. This yielded an Achromobacter xylosoxidans strain (Q19) that inactivated PQS, yielding a new fluorescent compound (I-PQS) confirmed as PQS-derived using deuterated PQS. The I-PQS structure was elucidated using mass spectrometry and nuclear magnetic resonance spectroscopy as 2-heptyl-2-hydroxy-1,2-dihydroquinoline- 3,4-dione (HHQD). Achromobacter xylosoxidans Q19 oxidized PQS congeners with alkyl chains ranging from C1 to C5 and also N-methyl PQS, yielding the corresponding 2-hydroxy-1,2-dihydroquinoline-3,4- diones, but was unable to inactivate thePQSprecursor HHQ. This indicates that the hydroxyl group at position 3 in PQS is essential and that A. xylosoxidans inactivates PQS via a pathway involving the incorporation of oxygen at C2 of the heterocyclic ring. The conversion of PQS to HHQD also occurred on incubation with 12/17 A. xylosoxidans strains recovered from cystic fibrosis patients, with P. aeruginosa and with Arthrobacter, suggesting that formation of hydroxylated PQS may be a common mechanism of inactivation

Biotic inactivation of the Pseudomonas aeruginosa quinolone signal molecule

In Pseudomonas aeruginosa, quorum sensing (QS) regulates the production of secondary metabolites, many of which are antimicrobials that impact on polymicrobial community composition. Consequently, quenching QS modulates the environmental impact of P. aeruginosa. To identify bacteria capable of inactivating the QS signal molecule 2-heptyl-3- hydroxy-4(1H)-quinolone (PQS), a minimal medium containing PQS as the sole carbon source was used to enrich a Malaysian rainforest soil sample. This yielded an Achromobacter xylosoxidans strain (Q19) that inactivated PQS, yielding a new fluorescent compound (I-PQS) confirmed as PQS-derived using deuterated PQS. The I-PQS structure was elucidated using mass spectrometry and nuclear magnetic resonance spectroscopy as 2-heptyl-2-hydroxy-1,2-dihydroquinoline- 3,4-dione (HHQD). Achromobacter xylosoxidans Q19 oxidized PQS congeners with alkyl chains ranging from C1 to C5 and also N-methyl PQS, yielding the corresponding 2-hydroxy-1,2-dihydroquinoline-3,4- diones, but was unable to inactivate thePQSprecursor HHQ. This indicates that the hydroxyl group at position 3 in PQS is essential and that A. xylosoxidans inactivates PQS via a pathway involving the incorporation of oxygen at C2 of the heterocyclic ring. The conversion of PQS to HHQD also occurred on incubation with 12/17 A. xylosoxidans strains recovered from cystic fibrosis patients, with P. aeruginosa and with Arthrobacter, suggesting that formation of hydroxylated PQS may be a common mechanism of inactivation

Enzyme-Mediated Quenching of the Pseudomonas Quinolone Signal (PQS) Promotes Biofilm Formation of Pseudomonas aeruginosa by Increasing Iron Availability

The 2-alkyl-3-hydroxy-4(1H)-quinolone 2,4-dioxygenase HodC was previously described to cleave the Pseudomonas quinolone signal, PQS, which is exclusively used in the complex quorum sensing (QS) system of Pseudomonas aeruginosa, an opportunistic pathogen employing QS to regulate virulence and biofilm development. Degradation of PQS by exogenous addition of HodC to planktonic cells of P.aeruginosa attenuated production of virulence factors, and reduced virulence inplanta. However, proteolytic cleavager educed the efficacy of HodC. Here, we identified the secreted protease LasB of P.aeruginosa to be responsible for HodC degradation. In static biofilms of the P.aeruginosa PA14 lasB::Tn mutant, the catalytic activity of HodC led to an increase in viable biomass in newly formed but also in established biofilms, and reduced the expression of genes involved in iron metabolism and siderophore production, such as pvdS, pvdL, pvdA, and pvdQ. This is likely due to an increase in the levels of bioavailable iron by degradation of PQS, which is able to sequester iron from the surrounding environment. Thus, HodC, despite ist ability to quench the production of virulence factors, is contraindicated for combating P.aeruginosa biofilms

PqsBC, a condensing enzyme in the biosynthesis of the Pseudomonas aeruginosa quinolone signal: crystal structure, inhibition, and reaction mechanism

Pseudomonas aeruginosa produces a number of alkylquinolone-type secondary metabolites best known for their antimicrobial effects and involvement in cell-cell communication. In the alkylquinolone biosynthetic pathway, the β-ketoacyl-(acyl carrier protein) synthase III (FabH)-like enzyme PqsBC catalyzes the condensation of octanoyl-coenzyme A and 2-aminobenzoylacetate (2-ABA) to form the signal molecule 2-heptyl-4(1H)-quinolone. PqsBC, a potential drug target, is unique for its heterodimeric arrangement and an active site different from that of canonical FabH-like enzymes. Considering the sequence dissimilarity between the subunits, a key question was how the two subunits are organized with respect to the active site. In this study, the PqsBC structure was determined to a 2 Å resolution, revealing that PqsB and PqsC have a pseudo-2-fold symmetry that unexpectedly mimics the FabH homodimer. PqsC has an active site composed of Cys-129 and His-269, and the surrounding active site cleft is hydrophobic in character and approximately twice the volume of related FabH enzymes that may be a requirement to accommodate the aromatic substrate 2-ABA. From physiological and kinetic studies, we identified 2-aminoacetophenone as a pathway-inherent competitive inhibitor of PqsBC, whose fluorescence properties could be used for in vitro binding studies. In a time-resolved setup, we demonstrated that the catalytic histidine is not involved in acyl-enzyme formation, but contributes to an acylation-dependent increase in affinity for the second substrate 2-ABA. Introduction of Asn into the PqsC active site led to significant activity toward the desamino substrate analog benzoylacetate, suggesting that the substrate 2-ABA itself supplies the asparagine-equivalent amino function that assists in catalysis