ABSTRACT
Pseudomonas aeruginosa forms suspended multicellular aggregates when cultured in liquid media. These aggregates may be important in disease, and/or as a pathway to biofilm formation. The polysaccharide Psl and extracellular DNA (eDNA) have both been implicated in aggregation, but previous results depend strongly on the experimental conditions. Here we develop a quantitative microscopy-based method for assessing changes in the size distribution of suspended aggregates over time in growing cultures. For exponentially growing cultures of P. aeruginosa PAO1, we find that aggregation is mediated by cell-associated Psl, rather than by either eDNA or secreted Psl. These aggregates arise de novo within the culture via a growth process that involves both collisions and clonal growth, and Psl non-producing cells do not aggregate with producers. In contrast, we find that stationary phase (overnight) cultures contain a different type of multicellular aggregate, in which both eDNA and Psl mediate cohesion. Our findings suggest that the physical and biological properties of multicellular aggregates may be very different in early-stage vs late-stage bacterial cultures.
Subject(s)
Biofilms , Pseudomonas aeruginosa , Polysaccharides, Bacterial/metabolism , DNAABSTRACT
Biosynthesis of the Pel exopolysaccharide in Pseudomonas aeruginosa requires all seven genes of the pelABCDEFG operon. The periplasmic modification enzyme PelA contains a C-terminal deacetylase domain that is necessary for Pel-dependent biofilm formation. Herein, we show that extracellular Pel is not produced by a P. aeruginosa PelA deacetylase mutant. This positions PelA deacetylase activity as an attractive target to prevent Pel-dependent biofilm formation. Using a high-throughput screen (n = 69,360), we identified 56 compounds that potentially inhibit PelA esterase activity, the first enzymatic step in the deacetylase reaction. A secondary biofilm inhibition assay identified methyl 2-(2-pyridinylmethylene) hydrazinecarbodithioate (SK-017154-O) as a specific Pel-dependent biofilm inhibitor. Structure-activity relationship studies identified the thiocarbazate as a necessary functional group and that the pyridyl ring could be replaced with a phenyl substituent (compound 1). Both SK-017154-O and compound 1 inhibit Pel-dependent biofilm formation in Bacillus cereus ATCC 10987, which has a predicted extracellular PelA deacetylase in its pel operon. Michaelis-Menten kinetics determined SK-017154-O to be a noncompetitive inhibitor of PelA, while compound 1 did not directly inhibit PelA esterase activity. Cytotoxicity assays using human lung fibroblast cells showed that compound 1 is less cytotoxic than SK-017154-O. This work provides proof of concept that biofilm exopolysaccharide modification enzymes are important for biofilm formation and can serve as useful antibiofilm targets. IMPORTANCE Present in more than 500 diverse Gram-negative and 900 Gram-positive organisms, the Pel polysaccharide is one of the most phylogenetically widespread biofilm matrix determinants found to date. Partial de-N-acetylation of this α-1,4 linked N-acetylgalactosamine polymer by the carbohydrate modification enzyme PelA is required for Pel-dependent biofilm formation in Pseudomonas aeruginosa and Bacillus cereus. Given this and our observation that extracellular Pel is not produced by a P. aeruginosa PelA deactylase mutant, we developed an enzyme-based high-throughput screen and identified methyl 2-(2-pyridinylmethylene) hydrazinecarbodithioate (SK-017154-O) and its phenyl derivative as specific Pel-dependent biofilm inhibitors. Michaelis-Menten kinetics revealed SK-017154-O is a noncompetitive inhibitor and that its noncytotoxic, phenyl derivative does not directly inhibit P. aeruginosa PelA esterase activity. We provide proof of concept that exopolysaccharide modification enzymes can be targeted with small molecule inhibitors to block Pel-dependent biofilm development in both Gram-negative and Gram-positive bacteria.
Subject(s)
Polysaccharides, Bacterial , Pseudomonas aeruginosa , Humans , Pseudomonas aeruginosa/genetics , Biofilms , Periplasm , Esterases , Bacterial Proteins/geneticsABSTRACT
The bacterium Pseudomonas aeruginosa can colonize the airways of patients with chronic lung disease. Within the lung, P. aeruginosa forms biofilms that can enhance resistance to antibiotics and immune defenses. P. aeruginosa biofilm formation is dependent on the secretion of matrix exopolysaccharides, including Pel and Psl. In this study, recombinant glycoside hydrolases (GHs) that degrade Pel and Psl were evaluated alone and in combination with antibiotics in a mouse model of P. aeruginosa infection. Intratracheal GH administration was well tolerated by mice. Pharmacokinetic analysis revealed that, although GHs have short half-lives, administration of two GHs in combination resulted in increased GH persistence. Combining GH prophylaxis and treatment with the antibiotic ciprofloxacin resulted in greater reduction in pulmonary bacterial burden than that with either agent alone. This study lays the foundation for further exploration of GH therapy in bacterial infections.
Subject(s)
Pseudomonas Infections , Animals , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Biofilms , Glycoside Hydrolases/metabolism , Lung/metabolism , Mice , Polysaccharides, Bacterial/metabolism , Pseudomonas Infections/drug therapy , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/metabolismABSTRACT
Aspergillus fumigatus is a ubiquitous mold that can cause invasive pulmonary infections in immunocompromised patients. Within the lung, A. fumigatus forms biofilms that can enhance resistance to antifungals and immune defenses. Aspergillus biofilm formation requires the production of a cationic matrix exopolysaccharide, galactosaminogalactan (GAG). In this study, recombinant glycoside hydrolases (GH)s that degrade GAG were evaluated as antifungal agents in a mouse model of invasive aspergillosis. Intratracheal GH administration was well tolerated by mice. Pharmacokinetic analysis revealed that although GHs have short half-lives, GH prophylaxis resulted in reduced fungal burden in leukopenic mice and improved survival in neutropenic mice, possibly through augmenting pulmonary neutrophil recruitment. Combining GH prophylaxis with posaconazole treatment resulted in a greater reduction in fungal burden than either agent alone. This study lays the foundation for further exploration of GH therapy in invasive fungal infections. IMPORTANCE The biofilm-forming mold Aspergillus fumigatus is a common causative agent of invasive fungal airway disease in patients with a compromised immune system or chronic airway disease. Treatment of A. fumigatus infection is limited by the few available antifungals to which fungal resistance is becoming increasingly common. The high mortality rate of A. fumigatus-related infection reflects a need for the development of novel therapeutic strategies. The fungal biofilm matrix is in part composed of the adhesive exopolysaccharide galactosaminogalactan, against which antifungals are less effective. Previously, we demonstrated antibiofilm activity with recombinant forms of the glycoside hydrolase enzymes that are involved in galactosaminogalactan biosynthesis. In this study, prophylaxis with glycoside hydrolases alone or in combination with the antifungal posaconazole in a mouse model of experimental aspergillosis improved outcomes. This study offers insight into the therapeutic potential of combining biofilm disruptive agents to leverage the activity of currently available antifungals.
Subject(s)
Antifungal Agents/administration & dosage , Aspergillus fumigatus/pathogenicity , Biofilms/drug effects , Glycoside Hydrolases/administration & dosage , Glycoside Hydrolases/genetics , Invasive Pulmonary Aspergillosis/prevention & control , Animals , Antifungal Agents/pharmacokinetics , Biofilms/growth & development , Disease Models, Animal , Drug Evaluation, Preclinical , Female , Glycoside Hydrolases/pharmacokinetics , Invasive Pulmonary Aspergillosis/microbiology , Mice , Mice, Inbred BALB C , Neutropenia , Recombinant Proteins/administration & dosage , Recombinant Proteins/genetics , VirulenceABSTRACT
During infection, the fungal pathogen Aspergillus fumigatus forms biofilms that enhance its resistance to antimicrobials and host defenses. An integral component of the biofilm matrix is galactosaminogalactan (GAG), a cationic polymer of α-1,4-linked galactose and partially deacetylated N-acetylgalactosamine (GalNAc). Recent studies have shown that recombinant hydrolase domains from Sph3, an A. fumigatus glycoside hydrolase involved in GAG synthesis, and PelA, a multifunctional protein from Pseudomonas aeruginosa involved in Pel polysaccharide biosynthesis, can degrade GAG, disrupt A. fumigatus biofilms, and attenuate fungal virulence in a mouse model of invasive aspergillosis. The molecular mechanisms by which these enzymes disrupt biofilms have not been defined. We hypothesized that the hydrolase domains of Sph3 and PelA (Sph3h and PelAh, respectively) share structural and functional similarities given their ability to degrade GAG and disrupt A. fumigatus biofilms. MALDI-TOF enzymatic fingerprinting and NMR experiments revealed that both proteins are retaining endo-α-1,4-N-acetylgalactosaminidases with a minimal substrate size of seven residues. The crystal structure of PelAh was solved to 1.54 Å and structure alignment to Sph3h revealed that the enzymes share similar catalytic site residues. However, differences in the substrate-binding clefts result in distinct enzyme-substrate interactions. PelAh hydrolyzed partially deacetylated substrates better than Sph3h, a finding that agrees well with PelAh's highly electronegative binding cleft versus the neutral surface present in Sph3h Our insight into PelAh's structure and function necessitate the creation of a new glycoside hydrolase family, GH166, whose structural and mechanistic features, along with those of GH135 (Sph3), are reported here.
Subject(s)
Biofilms/drug effects , Glycoside Hydrolases/metabolism , Polysaccharide-Lyases/ultrastructure , Anti-Infective Agents/metabolism , Aspergillus fumigatus/metabolism , Biofilms/growth & development , Catalytic Domain , Fungal Proteins/metabolism , Fungi/metabolism , Glycoside Hydrolases/physiology , Hydrolysis , Polysaccharide-Lyases/metabolism , Polysaccharides/metabolism , Pseudomonas aeruginosa/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Substrate Specificity/physiology , VirulenceABSTRACT
Pseudomonas aeruginosa is an opportunistic, nosocomial bacterial pathogen that forms persistent infections due to the formation of protective communities, known as biofilms. Once the biofilm is formed, the bacteria embedded within it are recalcitrant to antimicrobial treatment and host immune defenses. Moreover, the presence of biofilms in wounds is correlated with chronic infection and delayed healing. The current standard of care for chronic wound infections typically involves physical disruption of the biofilm via debridement and subsequent antimicrobial treatment. The glycoside hydrolases PelAh and PslGh have been demonstrated in vitro to disrupt biofilm integrity through degradation of the key biofilm matrix exopolysaccharides Pel and Psl, respectively. Herein, we demonstrate that PslGh hydrolase therapy is a promising strategy for controlling P. aeruginosa wound infections. Hydrolase treatment of P. aeruginosa biofilms resulted in increased antibiotic efficacy and penetration into the biofilm. PslGh treatment of P. aeruginosa biofilms also improved innate immune activity leading to greater complement deposition, neutrophil phagocytosis, and neutrophil reactive oxygen species production. Furthermore, when P. aeruginosa-infected wounds were treated with a combination of PslGh and tobramycin, we observed an additive effect leading to greater bacterial clearance than treatments of tobramycin or PslGh alone. This study demonstrates that PelAh and PslGh have promising therapeutic potential and that PslGh may aid in the treatment of P. aeruginosa wound infections.
Subject(s)
Anti-Bacterial Agents/pharmacology , Glycoside Hydrolases/pharmacology , Immunity, Innate/drug effects , Pseudomonas aeruginosa/drug effects , Wound Infection/drug therapy , Animals , Biofilms/drug effects , Female , Humans , Mice , Mice, Inbred BALB C , Neutrophils/drug effects , Neutrophils/metabolism , Phagocytosis/drug effects , Pseudomonas Infections/drug therapy , Pseudomonas Infections/metabolism , Reactive Oxygen Species/metabolism , Swine , Tobramycin/pharmacology , Wound Infection/metabolismABSTRACT
Poly-ß(1,6)-N-acetyl-D-glucosamine (PNAG) is a major biofilm component of many pathogenic bacteria. The production, modification, and export of PNAG in Escherichia coli and Bordetella species require the protein products encoded by the pgaABCD operon. PgaB is a two-domain periplasmic protein that contains an N-terminal deacetylase domain and a C-terminal PNAG binding domain that is critical for export. However, the exact function of the PgaB C-terminal domain remains unclear. Herein, we show that the C-terminal domains of Bordetella bronchiseptica PgaB (PgaBBb) and E. coli PgaB (PgaBEc) function as glycoside hydrolases. These enzymes hydrolyze purified deacetylated PNAG (dPNAG) from Staphylococcus aureus, disrupt PNAG-dependent biofilms formed by Bordetella pertussis, Staphylococcus carnosus, Staphylococcus epidermidis, and E. coli, and potentiate bacterial killing by gentamicin. Furthermore, we found that PgaBBb was only able to hydrolyze PNAG produced in situ by the E. coli PgaCD synthase complex when an active deacetylase domain was present. Mass spectrometry analysis of the PgaB-hydrolyzed dPNAG substrate showed a GlcN-GlcNAc-GlcNAc motif at the new reducing end of detected fragments. Our 1.76 Å structure of the C-terminal domain of PgaBBb reveals a central cavity within an elongated surface groove that appears ideally suited to recognize the GlcN-GlcNAc-GlcNAc motif. The structure, in conjunction with molecular modeling and site directed mutagenesis led to the identification of the dPNAG binding subsites and D474 as the probable catalytic acid. This work expands the role of PgaB within the PNAG biosynthesis machinery, defines a new glycoside hydrolase family GH153, and identifies PgaB as a possible therapeutic agent for treating PNAG-dependent biofilm infections.
Subject(s)
Amidohydrolases/metabolism , Biofilms/growth & development , Bordetella/enzymology , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Glycoside Hydrolases/metabolism , beta-Glucans/chemistry , Acetylation , Amidohydrolases/chemistry , Bordetella/growth & development , Crystallography, X-Ray , Escherichia coli/growth & development , Escherichia coli Proteins/chemistry , Glycoside Hydrolases/chemistry , Operon , Protein Conformation , beta-Glucans/metabolismABSTRACT
Bacterial colonization and biofilm formation on surfaces are typically mediated by the deposition of exopolysaccharides and conditioning protein layers. Pseudomonas aeruginosa is a nosocomial opportunistic pathogen that utilizes strain-specific exopolysaccharides such as Psl, Pel or alginate for both initial surface attachment and biofilm formation. To generate surfaces that resist P. aeruginosa colonization, we covalently bound a Psl-specific glycoside hydrolase (PslGh) to several, chemically-distinct surfaces using amine functionalization (APTMS) and glutaraldehyde (GDA) linking. In situ quartz crystal microbalance (QCM) experiments and fluorescence microscopy demonstrated a complete lack of Psl adsorption on the PslGh-bound surfaces. Covalently-bound PslGh was also found to significantly reduce P. aeruginosa surface attachment and biofilm formation over extended growth periods (8 days). The PslGh surfaces showed a â¼99.9% (â¼3-log) reduction in surface associated bacteria compared to control (untreated) surfaces, or those treated with inactive enzyme. This work demonstrates a non-eluting 'bioactive' surface that specifically targets a mechanism of cell adhesion, and that surface-bound glycoside hydrolase can significantly reduce surface colonization of bacteria through local, continuous enzymatic degradation of exopolysaccharide (Psl). These results have significant implications for the surface design of medical devices to keep bacteria in a planktonic state, and therefore susceptible to antibiotics and antimicrobials.
Subject(s)
Biocompatible Materials/pharmacology , Biofilms/drug effects , Enzymes, Immobilized/pharmacology , Glycoside Hydrolases/pharmacology , Polysaccharides, Bacterial/metabolism , Pseudomonas aeruginosa/drug effects , Bacterial Adhesion/drug effects , Biocompatible Materials/chemistry , Biofilms/growth & development , Enzymes, Immobilized/chemistry , Glycoside Hydrolases/chemistry , Humans , Models, Molecular , Pseudomonas Infections/microbiology , Pseudomonas Infections/prevention & control , Pseudomonas aeruginosa/physiology , Surface PropertiesABSTRACT
Galactosaminogalactan and Pel are cationic heteropolysaccharides produced by the opportunistic pathogens Aspergillus fumigatus and Pseudomonas aeruginosa, respectively. These exopolysaccharides both contain 1,4-linked N-acetyl-d-galactosamine and play an important role in biofilm formation by these organisms. Proteins containing glycoside hydrolase domains have recently been identified within the biosynthetic pathway of each exopolysaccharide. Recombinant hydrolase domains from these proteins (Sph3h from A. fumigatus and PelAh from P. aeruginosa) were found to degrade their respective polysaccharides in vitro. We therefore hypothesized that these glycoside hydrolases could exhibit antibiofilm activity and, further, given the chemical similarity between galactosaminogalactan and Pel, that they might display cross-species activity. Treatment of A. fumigatus with Sph3h disrupted A. fumigatus biofilms with an EC50 of 0.4 nM. PelAh treatment also disrupted preformed A. fumigatus biofilms with EC50 values similar to those obtained for Sph3h In contrast, Sph3h was unable to disrupt P. aeruginosa Pel-based biofilms, despite being able to bind to the exopolysaccharide. Treatment of A. fumigatus hyphae with either Sph3h or PelAh significantly enhanced the activity of the antifungals posaconazole, amphotericin B, and caspofungin, likely through increasing antifungal penetration of hyphae. Both enzymes were noncytotoxic and protected A549 pulmonary epithelial cells from A. fumigatus-induced cell damage for up to 24 h. Intratracheal administration of Sph3h was well tolerated and reduced pulmonary fungal burden in a neutropenic mouse model of invasive aspergillosis. These findings suggest that glycoside hydrolases can exhibit activity against diverse microorganisms and may be useful as therapeutic agents by degrading biofilms and attenuating virulence.
Subject(s)
Aspergillosis/therapy , Aspergillus fumigatus/enzymology , Bacterial Proteins/chemistry , Biofilms , Glycoside Hydrolases/chemistry , Pseudomonas aeruginosa/enzymology , A549 Cells , Animals , Anti-Infective Agents/chemistry , Antifungal Agents/chemistry , Aspergillosis/microbiology , Female , Humans , Mice , Mice, Inbred BALB C , Microbial Sensitivity Tests , Polysaccharides/chemistry , Species Specificity , VirulenceABSTRACT
Bacterial biofilms present a significant medical challenge because they are recalcitrant to current therapeutic regimes. A key component of biofilm formation in the opportunistic human pathogen Pseudomonas aeruginosa is the biosynthesis of the exopolysaccharides Pel and Psl, which are involved in the formation and maintenance of the structural biofilm scaffold and protection against antimicrobials and host defenses. Given that the glycoside hydrolases PelAh and PslGh encoded in the pel and psl biosynthetic operons, respectively, are utilized for in vivo exopolysaccharide processing, we reasoned that these would provide specificity to target P. aeruginosa biofilms. Evaluating these enzymes as potential therapeutics, we demonstrate that these glycoside hydrolases selectively target and degrade the exopolysaccharide component of the biofilm matrix. PelAh and PslGh inhibit biofilm formation over a 24-hour period with a half maximal effective concentration (EC50) of 69.3 ± 1.2 and 4.1 ± 1.1 nM, respectively, and are capable of disrupting preexisting biofilms in 1 hour with EC50 of 35.7 ± 1.1 and 12.9 ± 1.1 nM, respectively. This treatment was effective against clinical and environmental P. aeruginosa isolates and reduced biofilm biomass by 58 to 94%. These noncytotoxic enzymes potentiated antibiotics because the addition of either enzyme to a sublethal concentration of colistin reduced viable bacterial counts by 2.5 orders of magnitude when used either prophylactically or on established 24-hour biofilms. In addition, PelAh was able to increase neutrophil killing by ~50%. This work illustrates the feasibility and benefits of using bacterial exopolysaccharide biosynthetic glycoside hydrolases to develop novel antibiofilm therapeutics.
Subject(s)
Biofilms/growth & development , Glycoside Hydrolases/metabolism , Polysaccharides, Bacterial/biosynthesis , Pseudomonas aeruginosa/physiology , Anti-Bacterial Agents/pharmacology , Biofilms/drug effects , Catalysis , Cytotoxicity, Immunologic/drug effects , Environmental Microbiology , Enzyme Activation , Glycoside Hydrolases/chemistry , Humans , Hydrolysis , Neutrophils/immunology , Neutrophils/microbiology , Protein Interaction Domains and Motifs , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/isolation & purificationABSTRACT
A key component of colonization, biofilm formation, and protection of the opportunistic human pathogen Pseudomonas aeruginosa is the biosynthesis of the exopolysaccharide Psl. Composed of a pentameric repeating unit of mannose, glucose, and rhamnose, the biosynthesis of Psl is proposed to occur via a Wzx/Wzy-dependent mechanism. Previous genetic studies have shown that the putative glycoside hydrolase PslG is essential for Psl biosynthesis. To understand the function of this protein, the apo-structure of the periplasmic domain of PslG (PslG(31-442)) and its complex with mannose were determined to 2.0 and 1.9 Å resolution, respectively. Despite a domain architecture and positioning of catalytic residues similar to those of other family 39 glycoside hydrolases, PslG(31-442) exhibits a unique 32-Å-long active site groove that is distinct from other structurally characterized family members. PslG formed a complex with two mannose monosaccharides in this groove, consistent with binding data obtained from intrinsic tryptophan fluorescence. PslG was able to catalyze the hydrolysis of surface-associated Psl, and this activity was abolished in a E165Q/E276Q double catalytic variant. Surprisingly, P. aeruginosa variants with these chromosomal mutations as well as a pslG deletion mutant were still capable of forming Psl biofilms. However, overexpression of PslG in a pslG deletion background impaired biofilm formation and resulted in less surface-associated Psl, suggesting that regulation of this enzyme is important during polysaccharide biosynthesis.
Subject(s)
Biofilms , Glycoside Hydrolases/metabolism , Polysaccharides/biosynthesis , Pseudomonas aeruginosa/enzymology , Carbohydrate Sequence , Glycoside Hydrolases/chemistry , Models, Molecular , Molecular Sequence Data , Structure-Activity Relationship , Subcellular Fractions/enzymologyABSTRACT
Aspergillus fumigatus is the most virulent species within the Aspergillus genus and causes invasive infections with high mortality rates. The exopolysaccharide galactosaminogalactan (GAG) contributes to the virulence of A. fumigatus. A co-regulated five-gene cluster has been identified and proposed to encode the proteins required for GAG biosynthesis. One of these genes, sph3, is predicted to encode a protein belonging to the spherulin 4 family, a protein family with no known function. Construction of an sph3-deficient mutant demonstrated that the gene is necessary for GAG production. To determine the role of Sph3 in GAG biosynthesis, we determined the structure of Aspergillus clavatus Sph3 to 1.25 Å. The structure revealed a (ß/α)8 fold, with similarities to glycoside hydrolase families 18, 27, and 84. Recombinant Sph3 displayed hydrolytic activity against both purified and cell wall-associated GAG. Structural and sequence alignments identified three conserved acidic residues, Asp-166, Glu-167, and Glu-222, that are located within the putative active site groove. In vitro and in vivo mutagenesis analysis demonstrated that all three residues are important for activity. Variants of Asp-166 yielded the greatest decrease in activity suggesting a role in catalysis. This work shows that Sph3 is a glycoside hydrolase essential for GAG production and defines a new glycoside hydrolase family, GH135.
Subject(s)
Aspergillus fumigatus/metabolism , Coccidioidin/chemistry , Fungal Proteins/chemistry , Glycoside Hydrolases/chemistry , Polysaccharides/biosynthesis , Amino Acid Sequence , Aspergillus fumigatus/enzymology , Aspergillus fumigatus/pathogenicity , Catalysis , Catalytic Domain , Cell Wall/enzymology , Coccidioidin/genetics , Coccidioidin/physiology , Conserved Sequence , Crystallography, X-Ray , Fungal Proteins/genetics , Fungal Proteins/physiology , Glycoside Hydrolases/genetics , Glycoside Hydrolases/physiology , Hydrolysis , Molecular Sequence Data , Mutation , Polysaccharides/genetics , Protein Conformation , Sequence AlignmentABSTRACT
The O-acetylation of polysaccharides is a common modification used by pathogenic organisms to protect against external forces. Pseudomonas aeruginosa secretes the anionic, O-acetylated exopolysaccharide alginate during chronic infection in the lungs of cystic fibrosis patients to form the major constituent of a protective biofilm matrix. Four proteins have been implicated in the O-acetylation of alginate, AlgIJF and AlgX. To probe the biological function of AlgJ, we determined its structure to 1.83 Å resolution. AlgJ is a SGNH hydrolase-like protein, which while structurally similar to the N-terminal domain of AlgX exhibits a distinctly different electrostatic surface potential. Consistent with other SGNH hydrolases, we identified a conserved catalytic triad composed of D190, H192 and S288 and demonstrated that AlgJ exhibits acetylesterase activity in vitro. Residues in the AlgJ signature motifs were found to form an extensive network of interactions that are critical for O-acetylation of alginate in vivo. Using two different electrospray ionization mass spectrometry (ESI-MS) assays we compared the abilities of AlgJ and AlgX to bind and acetylate alginate. Binding studies using defined length polymannuronic acid revealed that AlgJ exhibits either weak or no detectable polymer binding while AlgX binds polymannuronic acid specifically in a length-dependent manner. Additionally, AlgX was capable of utilizing the surrogate acetyl-donor 4-nitrophenyl acetate to catalyze the O-acetylation of polymannuronic acid. Our results, combined with previously published in vivo data, suggest that the annotated O-acetyltransferases AlgJ and AlgX have separate and distinct roles in O-acetylation. Our refined model for alginate acetylation places AlgX as the terminal acetlytransferase and provides a rationale for the variability in the number of proteins required for polysaccharide O-acetylation.
Subject(s)
Alginates/metabolism , Bacterial Proteins/metabolism , Pseudomonas aeruginosa/enzymology , Acetylation , Bacterial Proteins/chemistry , Base Sequence , Glucuronic Acid/metabolism , Hexuronic Acids/metabolism , Molecular Sequence Data , Protein Structure, QuaternaryABSTRACT
The exopolysaccharide alginate, produced by mucoid Pseudomonas aeruginosa in the lungs of cystic fibrosis patients, undergoes two different chemical modifications as it is synthesized that alter the properties of the polymer and hence the biofilm. One modification, acetylation, causes the cells in the biofilm to adhere better to lung epithelium, form microcolonies, and resist the effects of the host immune system and/or antibiotics. Alginate biosynthesis requires 12 proteins encoded by the algD operon, including AlgX, and although this protein is essential for polymer production, its exact role is unknown. In this study, we present the X-ray crystal structure of AlgX at 2.15 Å resolution. The structure reveals that AlgX is a two-domain protein, with an N-terminal domain with structural homology to members of the SGNH hydrolase superfamily and a C-terminal carbohydrate-binding module. A number of residues in the carbohydrate-binding module form a substrate recognition "pinch point" that we propose aids in alginate binding and orientation. Although the topology of the N-terminal domain deviates from canonical SGNH hydrolases, the residues that constitute the Ser-His-Asp catalytic triad characteristic of this family are structurally conserved. In vivo studies reveal that site-specific mutation of these residues results in non-acetylated alginate. This catalytic triad is also required for acetylesterase activity in vitro. Our data suggest that not only does AlgX protect the polymer as it passages through the periplasm but that it also plays a role in alginate acetylation. Our results provide the first structural insight for a wide group of closely related bacterial polysaccharide acetyltransferases.
Subject(s)
Alginates/metabolism , Bacterial Proteins/physiology , Pseudomonas aeruginosa/metabolism , Acetylation , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Crystallography, X-Ray , Glucuronic Acid/metabolism , Hexuronic Acids/metabolism , Models, Molecular , Mutation , Nuclear Magnetic Resonance, Biomolecular , Protein ConformationABSTRACT
The Pel polysaccharide serves as an intercellular adhesin for the formation and maintenance of biofilms in the opportunistic pathogen Pseudomonas aeruginosa. Pel biosynthesis requires the products of a seven-gene operon, pelA-pelG, all of which are necessary for Pel-dependent biofilm formation and Pel-related phenotypes. One of the genes, pelA, encodes a protein with a predicted polysaccharide deacetylase domain. In this work, the role of the putative deacetylase domain in Pel production was examined. We first established that purified recombinant PelA hydrolyzed the pseudosubstrate p-nitrophenyl acetate in vitro, and site-specific mutations of predicted deacetylase active-site residues reduced activity greater than 10-fold. Additionally, these mutants were deficient in Pel-dependent biofilm formation and wrinkly colony morphology in vivo. Subcellular fractionation experiments demonstrate that PelA localizes to both the membrane and periplasmic fractions. Finally, antiserum against the Pel polysaccharide was generated, and PelA deacetylase mutants do not produce Pel-reactive material. Taken together, these results suggest that the deacetylase activity of PelA is important for the production of the Pel polysaccharide.
Subject(s)
Bacterial Proteins/metabolism , Polysaccharides, Bacterial/biosynthesis , Pseudomonas aeruginosa/enzymology , Pseudomonas aeruginosa/metabolism , Bacterial Proteins/genetics , Cell Membrane/metabolism , Periplasm/metabolism , Protein Structure, Tertiary/genetics , Protein Structure, Tertiary/physiologyABSTRACT
BphJ, a nonphosphorylating acylating aldehyde dehydrogenase, catalyzes the conversion of aldehydes to form acyl-coenzyme A in the presence of NAD(+) and coenzyme A (CoA). The enzyme is structurally related to the nonacylating aldehyde dehydrogenases, aspartate-ß-semialdehyde dehydrogenase and phosphorylating glyceraldehyde-3-phosphate dehydrogenase. Cys-131 was identified as the catalytic thiol in BphJ, and pH profiles together with site-specific mutagenesis data demonstrated that the catalytic thiol is not activated by an aspartate residue, as previously proposed. In contrast to the wild-type enzyme that had similar specificities for two- or three-carbon aldehydes, an I195A variant was observed to have a 20-fold higher catalytic efficiency for butyraldehyde and pentaldehyde compared to the catalytic efficiency of the wild type toward its natural substrate, acetaldehyde. BphJ forms a heterotetrameric complex with the class II aldolase BphI that channels aldehydes produced in the aldol cleavage reaction to the dehydrogenase via a molecular tunnel. Replacement of Ile-171 and Ile-195 with bulkier amino acid residues resulted in no more than a 35% reduction in acetaldehyde channeling efficiency, showing that these residues are not critical in gating the exit of the channel. Likewise, the replacement of Asn-170 in BphJ with alanine and aspartate did not substantially alter aldehyde channeling efficiencies. Levels of activation of BphI by BphJ N170A, N170D, and I171A were reduced by ≥3-fold in the presence of NADH and ≥4.5-fold when BphJ was undergoing turnover, indicating that allosteric activation of the aldolase has been compromised in these variants. The results demonstrate that the dehydrogenase coordinates the catalytic activity of BphI through allostery rather than through aldehyde channeling.
Subject(s)
Aldehyde Dehydrogenase/metabolism , Burkholderia/enzymology , Fructose-Bisphosphate Aldolase/metabolism , Pyruvic Acid/metabolism , Acetaldehyde/metabolism , Aldehyde Dehydrogenase/chemistry , Aldehyde Dehydrogenase/genetics , Allosteric Regulation , Burkholderia/chemistry , Burkholderia/genetics , Burkholderia/metabolism , Fructose-Bisphosphate Aldolase/chemistry , Models, Molecular , Mutagenesis, Site-Directed , Protein Conformation , Substrate SpecificityABSTRACT
Bacterial aldolase-dehydrogenase complexes catalyze the last steps in the meta cleavage pathway of aromatic hydrocarbon degradation. The aldolase (TTHB246) and dehydrogenase (TTHB247) from Thermus thermophilus were separately expressed and purified from recombinant Escherichia coli. The aldolase forms a dimer, while the dehydrogenase is a monomer; these enzymes can form a stable tetrameric complex in vitro, consisting of two aldolase and two dehydrogenase subunits. Upon complex formation, the K(m) value of 4-hydroxy-2-oxopentanoate, the substrate of TTHB246, is decreased 4-fold while the K(m) of acetaldehyde, the substrate of TTHB247, is increased 3-fold. The k(cat) values of each enzyme were reduced by ~2-fold when they were in a complex. The half-life of TTHB247 at 50 °C increased by ~4-fold when it was in a complex with TTHB246. The acetaldehyde product from TTHB246 could be efficiently channelled directly to TTHB247, but the channeling efficiency for the larger propionaldehyde was ~40% lower. A single A324G substitution in TTHB246 increased the channeling efficiency of propionaldehyde to a value comparable to that of acetaldehyde. Stable and catalytically competent chimeric complexes could be formed between the T. thermophilus enzymes and the orthologous aldolase (BphI) and dehydrogenase (BphJ) from the biphenyl degradation pathway of Burkholderia xenovorans LB400. However, channeling efficiencies for acetaldehyde in these chimeric complexes were ~10%. Structural and sequence analysis suggests that interacting residues in the interface of the aldolase-dehydrogenase complex are highly conserved among homologues, but coevolution of partner enzymes is required to fine-tune this interaction to allow for efficient substrate channeling.
Subject(s)
Bacterial Proteins/chemistry , Fructose-Bisphosphate Aldolase/chemistry , Oxidoreductases/chemistry , Bacterial Proteins/metabolism , Binding Sites , Burkholderia/enzymology , Burkholderia/metabolism , Catalysis , Escherichia coli/genetics , Escherichia coli/metabolism , Fructose-Bisphosphate Aldolase/metabolism , Kinetics , Models, Molecular , Oxidoreductases/metabolism , Protein Conformation , Substrate Specificity , Thermus thermophilus/enzymology , Thermus thermophilus/metabolismABSTRACT
BphI, a pyruvate-specific class II aldolase, catalyzes the reversible carbon-carbon bond formation of 4-hydroxy-2-oxoacids up to eight carbons in length. During the aldol addition catalyzed by BphI, the S-configured stereogenic center at C4 is created via attack of a pyruvate enolate intermediate on the si face of the aldehyde carbonyl of acetaldehyde to form 4(S)-hydroxy-2-oxopentanoate. Replacement of a Leu-87 residue within the active site of the enzyme with polar asparagine and bulky tryptophan led to enzymes with no detectable aldolase activity. These variants retained decarboxylase activity for the smaller oxaloacetate substrate, which is not inhibited by excess 4-hydroxy-2-oxopentanoate, confirming the results from molecular modeling that Leu-87 interacts with the C4-methyl of 4(S)-hydroxy-2-oxoacids. Double variants L87N;Y290F and L87W;Y290F were constructed to enable the binding of 4(R)-hydroxy-2-oxoacids by relieving the steric hindrance between the 5-methyl group of these compounds and the hydroxyl substituent on the phenyl ring of Tyr-290. The resultant enzymes were shown to exclusively utilize only 4(R)- and not 4(S)-hydroxy-2-oxopentanoate as the substrate. Polarimetric analysis confirmed that the double variants are able to synthesize 4-hydroxy-2-oxoacids up to eight carbons in length, which were the opposite stereoisomer compared to those produced by the wild-type enzyme. Overall the k(cat)/K(m) values for pyruvate and aldehydes in the aldol addition reactions were affected ≤10-fold in the double variants relative to the wild-type enzyme. Thus, stereocomplementary class II pyruvate aldolases are now available to create chiral 4-hydroxy-2-oxoacid skeletons as synthons for organic reactions.
Subject(s)
Drug Design , Fructose-Bisphosphate Aldolase/chemistry , Fructose-Bisphosphate Aldolase/metabolism , Aldehydes/metabolism , Animals , Carboxy-Lyases/metabolism , Catalytic Domain , Kinetics , Models, Molecular , Pentanoic Acids/chemistry , Pentanoic Acids/metabolism , Saccharomyces cerevisiae/enzymology , Stereoisomerism , Substrate SpecificityABSTRACT
Enzymes that catalyze carbon-carbon bond formation can be exploited as biocatalyst for synthetic organic chemistry. However, natural enzymes frequently do not possess the required properties or specificities to catalyze industrially useful transformations. This mini-review describes recent work using knowledge-guided site-specific mutagenesis of key active site residues to alter substrate specificity, stereospecificity and reaction specificity of these enzymes. In addition, examples of de novo designed enzymes that catalyze C-C bond reactions not found in nature will be discussed.
ABSTRACT
BphI-BphJ, an aldolase-dehydrogenase complex from the polychlorinated biphenyls (PCBs) degradation pathway, cleaves 4-hydroxy-2-oxoacids to pyruvate and an aldehyde. The enzyme complex was shown to exhibit substrate channeling, whereby linear aldehydes of up to 6 carbons long and branched isobutyraldehyde were directly channeled from the aldolase to the dehydrogenase with greater than 80% efficiency. BphI variants G322F, G322L, and G323F were created and were found to block aldehyde channeling. The dehydrogenase cofactor NADH was able to activate the catalytic activity of the aldol cleavage reaction in these variants, suggesting that activation of BphI by BphJ cofactors is not solely due to faster aldehyde release. A G323L variant was able to channel acetaldehyde but not the larger propionaldehyde while the G323A variant was able to channel butyraldehyde but not its isomer isobutyraldehyde, confirming that the restricted channeling of aldehydes in these glycine variants are due to steric blockage of the channel. Substitution of His-20 and Tyr-290 in BphI led to significant reductions in aldehyde channeling efficiencies. A mechanism of substrate channeling involving these two gating residues is proposed.