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Pel exopolysaccharide biosynthetic loci are phylogenetically widespread biofilm matrix determinants in bacteria. In Pseudomonas aeruginosa, Pel is crucial for cell-to-cell interactions and reducing susceptibility to antibiotic and mucolytic treatments. While genes encoding glycoside hydrolases have long been linked to biofilm exopolysaccharide biosynthesis, their physiological role in biofilm development is unclear. Here we demonstrate that the glycoside hydrolase activity of P. aeruginosa PelA decreases adherent biofilm biomass and is responsible for generating the low molecular weight secreted form of the Pel exopolysaccharide. We show that the generation of secreted Pel contributes to the biomechanical properties of the biofilm and decreases the virulence of P. aeruginosa in Caenorhabditis elegans and Drosophila melanogaster. Our results reveal that glycoside hydrolases found in exopolysaccharide biosynthetic systems can help shape the soft matter attributes of a biofilm and propose that secreted matrix components be referred to as matrix associated to better reflect their influence., (© 2023. The Author(s).)

Synthase-dependent secretion systems are a conserved mechanism for producing exopolysaccharides in Gram-negative bacteria. Although widely studied, it is not well understood how these systems are organized to coordinate polymer biosynthesis, modification, and export across both membranes and the peptidoglycan. To investigate how synthase-dependent secretion systems produce polymer at a molecular level, we determined the crystal structure of the AlgK-AlgX (AlgKX) complex involved in Pseudomonas aeruginosa alginate exopolysaccharide acetylation and export. We demonstrate that AlgKX directly binds alginate oligosaccharides and that formation of the complex is vital for polymer production and biofilm attachment. Finally, we propose a structural model for the AlgEKX outer membrane modification and secretion complex. Together, our study provides insight into how alginate biosynthesis proteins coordinate production of a key exopolysaccharide involved in establishing persistent Pseudomonas lung infections., (© 2022. The Author(s).)

Bacteria require polysaccharides for structure, survival, and virulence. Despite their central role in microbiology, few tools are available to manipulate their production. In E. coli , the glycosyltransferase complex PgaCD produces poly- N -acetylglucosamine (PNAG), an extracellular matrix polysaccharide required for biofilm formation. We report that C6-substituted (H, F, N 3 , SH, NH 2 ) UDP-GlcNAc substrate analogues are inhibitors of PgaCD. In vitro , the inhibitors cause PNAG chain termination, consistent with the mechanism of PNAG polymerization from the nonreducing terminus. In vivo , expression of the GlcNAc-1-kinase NahK in E. coli provided a non-native GlcNAc salvage pathway that produced the UDP-GlcNAc analogue inhibitors in situ . The 6-fluoro and 6-deoxy derivatives were potent inhibitors of biofilm formation in the transformed strain, providing a tool to manipulate this key exopolysaccharide. Characterization of the UDP-GlcNAc pool and quantification of PNAG generation support PNAG termination as the primary in vivo mechanism of biofilm inhibition by 6-fluoro UDP-GlcNAc.

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.

Implanted medical devices such as central venous catheters are highly susceptible to microbial colonization and biofilm formation and are a major risk factor for nosocomial infections. The opportunistic pathogen Pseudomonas aeruginosa uses exopolysaccharides, such as Psl, for both initial surface attachment and biofilm formation. We have previously shown that chemically immobilizing the Psl-specific glycoside hydrolase, PslG h , to a material surface can inhibit P. aeruginosa biofilm formation. Herein, we show that PslG h can be uniformly immobilized on the lumen surface of medical-grade, commercial polyethylene, polyurethane, and polydimethylsiloxane (silicone) catheter tubing. We confirmed that the surface-bound PslG h was uniformly distributed along the catheter length and remained active even after storage for 30 days at 4 °C. P. aeruginosa colonization and biofilm formation under dynamic flow culture conditions in vitro showed a 3-log reduction in the number of bacteria during the first 11 days, and a 2-log reduction by day 14 for PslG h -modified PE-100 catheters, compared to untreated catheter controls. In an in vivo rat infection model, PslG h -modified PE-100 catheters showed a ∼1.5-log reduction in the colonization of the clinical P. aeruginosa ATCC 27853 strain after 24 h. These results demonstrate the robust ability of surface-bound glycoside hydrolase enzymes to inhibit biofilm formation and their potential to reduce rates of device-associated infections.

Biofilm-dispersing enzymes degrade the extracellular polymeric matrix surrounding bacterial biofilms, disperse the microbial community and increase their susceptibility to antibiotics and immune cells. Challenges for the clinical translation of biofilm-dispersing enzymes involve their susceptibility to denaturation, degradation, and clearance upon administration in vivo. Drug delivery systems aim to overcome these limitations through encapsulation, stabilization and protection from the exterior environment, thereby maintaining the enzymatic activity. Smart drug delivery systems offer target specificity, releasing payloads at the site of infection while minimizing unnecessary systemic exposure. This review highlights critical advances of biofilm-dispersing enzymes as a novel therapeutic approach for biofilm-associated infections. We explore how smart, bio-responsive delivery systems overcome the limiting factors of biofilm-dispersing enzymes and summarize the key systems designed. This review will guide future developments, focusing on utilizing selective and specific therapies in a targeted fashion to meet the unmet therapeutic needs of biofilm infections., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

The glycoside hydrolase, PslG, attacks and degrades the dominant Psl polysaccharide in the exopolymeric substance (EPS) matrix of Pseudomonas aeruginosa biofilms and is a promising therapy to potentiate the effect of antibiotics. However, the need for coadministration with an antibiotic and the potential susceptibility of PslG to proteolysis highlights the need for an effective delivery system. Here, we compared liposomes versus lipid liquid crystal nanoparticles (LCNPs) loaded with PslG and tobramycin as potential formulation approaches to (1) protect PslG from proteolysis, (2) trigger the enzyme's release in the presence of bacteria, and (3) improve the total antimicrobial effect in vitro and in vivo in a Caenorhabditis elegans infection model. LCNPs were an effective formulation strategy for PslG and tobramycin that better protected the enzyme against proteolysis, triggered and sustained the release of PslG, improved the antimicrobial effect by 10-100-fold, and increased the survival of C. elegans infected with P. aeruginosa. Digestible LCNPs had the advantage of triggering the enzyme's release in the presence of bacteria. However, compared to nondigestible LCNPs, negligible differences arose between the LCNPs' ability to protect PslG from proteolysis and potentiate the antimicrobial activity in combination with tobramycin. In C. elegans , the improved antimicrobial efficacy was comparable to tobramycin-LCNPs, although the PslG + tobramycin-LCNPs achieved a greater than 10-fold reduction in bacteria compared to the unformulated combination. Herewith, LCNPs are showcased as a promising protective delivery system for novel biofilm dispersing enzymes combined with antibiotics, enabling infection-directed therapy and improved performance.

Bacteria synthesize and export adhesive macromolecules to enable biofilm formation. These macromolecules, collectively called the biofilm matrix, are structurally varied and often unique to specific bacterial species or subspecies. This heterogeneity in matrix utilization makes it difficult to facilitate direct comparison between biofilm formation mechanisms of different bacterial species. Despite this, some matrix components, in particular the polysaccharides poly-β-1,6- N -acetyl-glucosamine (PNAG) and bacterial cellulose, are utilized by many Gram-negative species for biofilm formation. However, there is a very narrow distribution of these components across Gram-positive organisms, whose biofilm matrix determinants remain largely undiscovered. We found that a genetic locus required for the production of a biofilm matrix component of P. aeruginosa , the Pel polysaccharide, is widespread in Gram-negative bacteria and that there is a variant form of this cluster present in many Gram-positive bacterial species. We demonstrated that this locus is required for biofilm formation by Bacillus cereus ATCC 10987, produces a polysaccharide that is similar to Pel, and is post-translationally regulated by cyclic-3',5'-dimeric-guanosine monophosphate (c-di-GMP) in a manner identical to P. aeruginosa . However, while the proposed mechanism for Pel production appears remarkably similar between B. cereus and P. aeruginosa , we identified several key differences between Gram-negative and Gram-positive Pel biosynthetic components in other monoderms. In particular, 4 different architectural subtypes of the c-di-GMP-binding component PelD were identified, including 1 found only in Streptococci that has entirely lost the c-di-GMP recognition domain. These observations highlight how existing multi-component bacterial machines can be subtly tweaked to adapt to the unique physiology and regulatory mechanisms of Gram-positive organisms. Collectively, our analyses suggest that the Pel biosynthetic locus is one of the most phylogenetically widespread biofilm matrix determinants in bacteria, and that its mechanism of production and regulation is extraordinarily conserved across the majority of organisms that possess it., Competing Interests: Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2021.)

The exopolysaccharide galactosaminogalactan (GAG) is an important virulence factor of the fungal pathogen Aspergillus fumigatus. Deletion of a gene encoding a putative deacetylase, Agd3, leads to defects in GAG deacetylation, biofilm formation, and virulence. Here, we show that Agd3 deacetylates GAG in a metal-dependent manner, and is the founding member of carbohydrate esterase family CE18. The active site is formed by four catalytic motifs that are essential for activity. The structure of Agd3 includes an elongated substrate-binding cleft formed by a carbohydrate binding module (CBM) that is the founding member of CBM family 87. Agd3 homologues are encoded in previously unidentified putative bacterial exopolysaccharide biosynthetic operons and in other fungal genomes.

Inhalation of conidia of the opportunistic mold Aspergillus fumigatus by immunocompromised hosts can lead to invasive pulmonary disease. Inhaled conidia that escape immune defenses germinate to form filamentous hyphae that invade lung tissues. Conidiation rarely occurs during invasive infection of the human host, allowing the bulk of fungal energy to be directed toward vegetative growth. We hypothesized that forced induction of conidiation during infection can suppress A. fumigatus vegetative growth, impairing the ability of this organism to cause disease. To study the effects of conidiation pathway dysregulation on A. fumigatus virulence, a key transcriptional regulator of conidiation ( brlA ) was expressed under the control of a doxycycline-inducible promoter. Time- and dose-dependent brlA overexpression was observed in response to doxycycline both in vitro and in vivo. Exposure of the inducible brlA overexpression strain to low doses of doxycycline under vegetative growth conditions in vitro induced conidiation, whereas high doses arrested growth. Overexpression of brlA attenuated A. fumigatus virulence in both an invertebrate and mouse model of invasive aspergillosis. RNA sequencing studies and phenotypic analysis revealed that brlA overexpression results in altered cell signaling, amino acid, and carbohydrate metabolism, including a marked upregulation of trehalose biosynthesis and a downregulation in the biosynthesis of the polysaccharide virulence factor galactosaminogalactan. This proof of concept study demonstrates that activation of the conidiation pathway in A. fumigatus can reduce virulence and suggests that brlA -inducing small molecules may hold promise as a new class of therapeutics for A. fumigatus infection. IMPORTANCE The mold Aspergillus fumigatus reproduces by the production of airborne spores (conidia), a process termed conidiation. In immunocompromised individuals, inhaled A. fumigatus conidia can germinate and form filaments that penetrate and damage lung tissues; however, conidiation does not occur during invasive infection. In this study, we demonstrate that forced activation of conidiation in filaments of A. fumigatus can arrest their growth and impair the ability of this fungus to cause disease in both an insect and a mouse model of invasive infection. Activation of conidiation was linked to profound changes in A. fumigatus metabolism, including a shift away from the synthesis of polysaccharides required for cell wall structure and virulence in favor of carbohydrates used for energy storage and stress resistance. Collectively, these findings suggest that activation of the conidiation pathway may be a promising approach for the development of new agents to prevent or treat A. fumigatus infection., (Copyright © 2020 Stewart et al.)

Type IV pilus-like systems are protein complexes that polymerize pilin fibres. They are critical for virulence in many bacterial pathogens. Pilin polymerization and depolymerization are powered by motor ATPases of the PilT/VirB11-like family. This family is thought to operate with C 2 symmetry; however, most of these ATPases crystallize with either C 3 or C 6 symmetric conformations. The relevance of these conformations is unclear. Here, we determine the X-ray structures of PilT in four unique conformations and use these structures to classify the conformation of available PilT/VirB11-like family member structures. Single particle electron cryomicroscopy (cryoEM) structures of PilT reveal condition-dependent preferences for C 2, C 3 , and C 6 conformations. The physiologic importance of these conformations is validated by coevolution analysis and functional studies of point mutants, identifying a rare gain-of-function mutation that favours the C 2 conformation. With these data, we propose a comprehensive model of PilT function with broad implications for PilT/VirB11-like family members.

Many clinically-relevant biofilm-forming bacterial strains produce partially de-N-acetylated poly-β-(1→6)-N-acetyl-d-glucosamine (dPNAG) as an exopolysaccharide. In Gram-negative bacteria, the periplasmic protein PgaB is responsible for partial de-N-acetylation of PNAG prior to its export to the extracellular space. In addition to de-N-acetylase activity found in the N-terminal domain, PgaB contains a C-terminal hydrolase domain that can disrupt dPNAG-dependent biofilms and hydrolyzes dPNAG but not fully acetylated PNAG. The role of this C-terminal domain in biofilm formation has yet to be determined in vivo. Further characterization of the enzyme's hydrolase activity has been hampered by a lack of specific dPNAG oligosaccharides. Here, we report the synthesis of a defined mono de-N-acetylated dPNAG penta- and hepta-saccharide. Using mass spectrometry analysis and a fluorescence-based thin-layer chromatography (TLC) assay, we found that our defined dPNAG oligosaccharides are hydrolase substrates. In addition to the expected cleavage site, two residues to the reducing side of the de-N-acetylated residue, minor cleavage products on the non-reducing side of the de-N-acetylation site were observed. These findings provide quantitative data to support how PNAG is processed in Gram-negative bacteria.

Aspergillus fumigatus is an opportunistic fungal pathogen that causes both chronic and acute invasive infections. Galactosaminogalactan (GAG) is an integral component of the A. fumigatus biofilm matrix and a key virulence factor. GAG is a heterogeneous linear α-1,4-linked exopolysaccharide of galactose and GalNAc that is partially deacetylated after secretion. A cluster of five co-expressed genes has been linked to GAG biosynthesis and modification. One gene in this cluster, ega3, is annotated as encoding a putative α-1,4-galactosaminidase belonging to glycoside hydrolase family 114 (GH114). Herein, we show that recombinant Ega3 is an active glycoside hydrolase that disrupts GAG-dependent A. fumigatus and Pel polysaccharide-dependent Pseudomonas aeruginosa biofilms at nanomolar concentrations. Using MS and functional assays, we demonstrate that Ega3 is an endo-acting α-1,4-galactosaminidase whose activity depends on the conserved acidic residues, Asp-189 and Glu-247. X-ray crystallographic structural analysis of the apo Ega3 and an Ega3-galactosamine complex, at 1.76 and 2.09 Å resolutions, revealed a modified (β/α) 8 -fold with a deep electronegative cleft, which upon ligand binding is capped to form a tunnel. Our structural analysis coupled with in silico docking studies also uncovered the molecular determinants for galactosamine specificity and substrate binding at the -2 to +1 binding subsites. The findings in this study increase the structural and mechanistic understanding of the GH114 family, which has >600 members encoded by plant and opportunistic human pathogens, as well as in industrially used bacteria and fungi., (© 2019 Bamford et al.)

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 PelA h and PslG h 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 PslG h 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. PslG h 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 PslG h and tobramycin, we observed an additive effect leading to greater bacterial clearance than treatments of tobramycin or PslG h alone. This study demonstrates that PelA h and PslG h have promising therapeutic potential and that PslG h may aid in the treatment of P. aeruginosa wound infections., (Copyright © 2019 American Society for Microbiology.)

Type IV pilus (T4P)-like systems have been identified in almost every major phylum of prokaryotic life. They include the type IVa pilus (T4aP), type II secretion system (T2SS), type IVb pilus (T4bP), Tad/Flp pilus, Com pilus, and archaeal flagellum (archaellum). These systems are used for adhesion, natural competence, phage adsorption, folded-protein secretion, surface sensing, swimming motility, and twitching motility. The T4aP allows for all of these functions except swimming and is therefore a good model system for understanding T4P-like systems. Recent structural analyses have revolutionized our understanding of how the T4aP machinery assembles and functions. Here we review the structure and function of the T4aP.

Competing Interests: The authors have declared that no competing interests exist.

Bacteria predominantly exist as matrix embedded communities of cells called biofilms. The biofilm matrix is made up of a variety of self-produced extracellular components including DNA, proteins, and exopolysaccharides. Bacterial exopolysaccharides have been implicated in surface adhesion, resistance to antibiotics, and protection from host immune systems. Herein we review the structure and function of the proteins involved in the production of the Gram-negative synthase-dependent exopolysaccharides: alginate, poly-β(1,6)-N-acetyl-d-glucosamine (PNAG), cellulose, and the Pel polysaccharide. We highlight the similarities and differences that exist at the molecular level in these synthase systems., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Bacterial surface (S) layers are paracrystalline arrays of protein assembled on the bacterial cell wall that serve as protective barriers and scaffolds for housekeeping enzymes and virulence factors. The attachment of S-layer proteins to the cell walls of the Bacillus cereus sensu lato, which includes the pathogen Bacillus anthracis, occurs through noncovalent interactions between their S-layer homology domains and secondary cell wall polysaccharides. To promote these interactions, it is presumed that the terminal N-acetylmannosamine (ManNAc) residues of the secondary cell wall polysaccharides must be ketal-pyruvylated. For a few specific S-layer proteins, the O-acetylation of the penultimate N-acetylglucosamine (GlcNAc) is also required. Herein, we present the X-ray crystal structure of the SLH domain of the major surface array protein Sap from B. anthracis in complex with 4,6- O-ketal-pyruvyl-β-ManNAc-(1,4)-β-GlcNAc-(1,6)-α-GlcN. This structure reveals for the first time that the conserved terminal SCWP unit is the direct ligand for the SLH domain. Furthermore, we identify key binding interactions that account for the requirement of 4,6- O-ketal-pyruvyl-ManNAc while revealing the insignificance of the O-acetylation on the GlcNAc residue for recognition by Sap.

O-Acetylation of the secondary cell wall polysaccharides (SCWP) of the Bacillus cereus group of pathogens, which includes Bacillus anthracis, is essential for the proper attachment of surface-layer (S-layer) proteins to their cell walls. Using a variety of pseudosubstrates and a chemically synthesized analog of SCWP, we report here the identification of PatB1 as a SCWP O-acetyltransferase in Bacillus cereus. Additionally, we report the crystal structure of PatB1, which provides detailed insights into the mechanism of this enzyme and defines a novel subfamily of the SGNH family of esterases and lipases. We propose a model for the O-acetylation of SCWP requiring the translocation of acetyl groups from a cytoplasmic source across the plasma membrane by PatA1 and PatA2 for their transfer to SCWP by PatB1.

The pellicle (PEL) polysaccharide is synthesized by the opportunistic pathogen Pseudomonas aeruginosa and is an important biofilm constituent critical for bacterial virulence and persistence. PEL is a cationic polymer that promotes cell-cell interactions within the biofilm matrix through electrostatic interactions with extracellular DNA. Translocation of PEL across the outer membrane is proposed to occur via PelB, a membrane-embedded porin with a large periplasmic domain predicted to contain 19 tetratricopeptide repeats (TPRs). TPR-containing domains are typically involved in protein-protein interactions, and we therefore sought to determine whether PelB serves as a periplasmic scaffold that recruits other components of the PEL secretion apparatus. In this study, we show that the TPR domain of PelB interacts with PelA, an enzyme with PEL deacetylase and hydrolase activities. Structure determination of PelB TPRs 8-11 enabled us to design systematic deletions of individual TPRs and revealed that repeats 9-14, which are required for the cellular localization of PelA with PelB are also essential for PEL-dependent biofilm formation. Copurification experiments indicated that the interaction between PelA and PelB is direct and that the deacetylase activity of PelA increases and its hydrolase activity decreases when these proteins interact. Combined, our results indicate that the TPR-containing domain of PelB localizes PelA to the PEL secretion apparatus within the periplasm and that this may allow for efficient deacetylation of PEL before its export from the cell., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Described is the first synthesis of guanosine diphosphate mannuronic acid (GDP-ManA), the sugar donor used by algae and bacteria for the production of alginate, an anionic polysaccharide composed of β-d-mannuronic acid (ManA) and α-l-guluronic acid (GulA). Understanding the biosynthesis of these polyanionic polysaccharides on the molecular level, opens up avenues to use and modulate the biosynthesis machinery for biotechnological and therapeutic applications. The synthesis reported here delivers multi-milligram amounts of the GDP-ManA donor that can be used to study the polymerase (Alg8 in Pseudomonas aeruginosa) that generates the poly-ManA chain. Also reported is the assembly of two close analogues of GDP-ManA: the first bears a C-4-O-methyl group, while the second has been deoxygenated at this position. Both molecules may be used as "chain stoppers" in future enzymatic ManA polymerisation reactions. The crucial pyrophosphate linkage of the GDP-mannuronic acids has been constructed by the phosphorylation of the appropriate ManA-1-phosphates with a guanosine phosphoramidite., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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.

Allelic exchange is an efficient method of bacterial genome engineering. This protocol describes the use of this technique to make gene knockouts and knock-ins, as well as single-nucleotide insertions, deletions and substitutions, in Pseudomonas aeruginosa. Unlike other approaches to allelic exchange, this protocol does not require heterologous recombinases to insert or excise selective markers from the target chromosome. Rather, positive and negative selections are enabled solely by suicide vector-encoded functions and host cell proteins. Here, mutant alleles, which are flanked by regions of homology to the recipient chromosome, are synthesized in vitro and then cloned into allelic exchange vectors using standard procedures. These suicide vectors are then introduced into recipient cells by conjugation. Homologous recombination then results in antibiotic-resistant single-crossover mutants in which the plasmid has integrated site-specifically into the chromosome. Subsequently, unmarked double-crossover mutants are isolated directly using sucrose-mediated counter-selection. This two-step process yields seamless mutations that are precise to a single base pair of DNA. The entire procedure requires ∼2 weeks.

Exopolysaccharides are required for the development and integrity of biofilms produced by a wide variety of bacteria. In Escherichia coli, partial de-N-acetylation of the exopolysaccharide poly-β-1,6-N-acetyl-D-glucosamine (PNAG) by the periplasmic protein PgaB is required for polysaccharide intercellular adhesin-dependent biofilm formation. To understand the molecular basis for PNAG de-N-acetylation, the structure of PgaB in complex with Ni(2+) and Fe(3+) have been determined to 1.9 and 2.1 Å resolution, respectively, and its activity on β-1,6-GlcNAc oligomers has been characterized. The structure of PgaB reveals two (β/α)(x) barrel domains: a metal-binding de-N-acetylase that is a member of the family 4 carbohydrate esterases (CE4s) and a domain structurally similar to glycoside hydrolases. PgaB displays de-N-acetylase activity on β-1,6-GlcNAc oligomers but not on the β-1,4-(GlcNAc)(4) oligomer chitotetraose and is the first CE4 member to exhibit this substrate specificity. De-N-acetylation occurs in a length-dependent manor, and specificity is observed for the position of de-N-acetylation. A key aspartic acid involved in de-N-acetylation, normally seen in other CE4s, is missing in PgaB, suggesting that the activity of PgaB is attenuated to maintain the low levels of de-N-acetylation of PNAG observed in vivo. The metal dependence of PgaB is different from most CE4s, because PgaB shows increased rates of de-N-acetylation with Co(2+) and Ni(2+) under aerobic conditions, and Co(2+), Ni(2+) and Fe(2+) under anaerobic conditions, but decreased activity with Zn(2+). The work presented herein will guide inhibitor design to combat biofilm formation by E. coli and potentially a wide range of medically relevant bacteria producing polysaccharide intercellular adhesin-dependent biofilms.

Secretins are channels that allow translocation of macromolecules across the outer membranes of Gram-negative bacteria. Virulence, natural competence, and motility are among the functions mediated by these large oligomeric protein assemblies. Filamentous phage also uses secretins to exit their bacterial host without causing cell lysis. However, the secretin is only a part of a larger membrane-spanning complex, and additional proteins are often required for its formation. A class of outer membrane lipoproteins called pilotins has been implicated in secretin assembly and/or localization. Additional accessory proteins may also be involved in secretin stability. Significant progress has recently been made toward deciphering the complex interactions required for functional secretin assembly. To allow for easier comparison between different systems, we have classified the secretins into five different classes based on their requirements for proteins involved in their assembly, localization, and stability. An overview of pilotin and accessory protein structures, functions, and characterized modes of interaction with the secretin is presented., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The production of the PEL polysaccharide in Pseudomonas aeruginosa requires the binding of bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) to the cytoplasmic GGDEF domain of the inner membrane protein PelD. Here, the overexpression, purification and crystallization of a soluble construct of PelD that encompasses the GGDEF domain and a predicted GAF domain is reported. Diffraction-quality crystals were grown using the hanging-drop vapour-diffusion method. The crystals grew as flat plates, with unit-cell parameters a = 88.3, b = 114.0, c = 61.9 Å, α = β = γ = 90.0°. The PelD crystals exhibited the symmetry of space group P2(1)2(1)2 and diffracted to a minimum d-spacing of 2.2 Å. On the basis of the Matthews coefficient (V(M) = 2.29 Å(3) Da(-1)), it was estimated that two molecules are present in the asymmetric unit.

Type IV pili (T4P) are retractile appendages that contribute to the virulence of bacterial pathogens. PilF is a Pseudomonas aeruginosa lipoprotein that is essential for T4P biogenesis. Phenotypic characterization of a pilF mutant confirmed that T4P-mediated functions are abrogated: T4P were no longer present on the cell surface, twitching motility was abolished, and the mutant was resistant to infection by T4P retraction-dependent bacteriophage. The results of cellular fractionation studies indicated that PilF is the outer membrane pilotin required for the localization and multimerization of the secretin, PilQ. Mutation of the putative PilF lipidation site untethered the protein from the outer membrane, causing secretin assembly in both inner and outer membranes. T4P-mediated twitching motility and bacteriophage susceptibility were moderately decreased in the lipidation site mutant, while cell surface piliation was substantially reduced. The tethering of PilF to the outer membrane promotes the correct localization of PilQ and appears to be required for the formation of stable T4P. Our 2.0-A structure of PilF revealed a superhelical arrangement of six tetratricopeptide protein-protein interaction motifs that may mediate the contacts with PilQ during secretin assembly. An alignment of pseudomonad PilF sequences revealed three highly conserved surfaces that may be involved in PilF function.

Distributed replica sampling [T. Rodinger et al., J. Chem. Theory Comput. 2, 725 (2006)] is a simple and general scheme for Boltzmann sampling of conformational space by computer simulation in which multiple replicas of the system undergo a random walk in reaction coordinate or temperature space. Individual replicas are linked through a generalized Hamiltonian containing an extra potential energy term or bias which depends on the distribution of all replicas, thus enforcing the desired sampling distribution along the coordinate or parameter of interest regardless of free energy barriers. In contrast to replica exchange methods, efficient implementation of the algorithm does not require synchronicity of the individual simulations. The algorithm is inherently suited for large-scale simulations using shared or heterogeneous computing platforms such as a distributed network. In this work, we build on our original algorithm by introducing Boltzmann-weighted jumping, which allows moves of a larger magnitude and thus enhances sampling efficiency along the reaction coordinate. The approach is demonstrated using a realistic and biologically relevant application; we calculate the standard binding free energy of benzene to the L99A mutant of T4 lysozyme. Distributed replica sampling is used in conjunction with thermodynamic integration to compute the potential of mean force for extracting the ligand from protein and solvent along a nonphysical spatial coordinate. Dynamic treatment of the reaction coordinate leads to faster statistical convergence of the potential of mean force than a conventional static coordinate, which suffers from slow transitions on a rugged potential energy surface.

5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes the irreversible cleavage of the glycosidic bond in 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH) and plays a key role in four metabolic processes: biological methylation, polyamine biosynthesis, methionine recycling and bacterial quorum sensing. The absence of the nucleosidase in mammalian species has implicated this enzyme as a target for antimicrobial drug design. MTAN from the pathogenic bacterium Staphylococcus aureus (SaMTAN) has been kinetically characterized and its structure has been determined in complex with the transition-state analogue formycin A (FMA) at 1.7 A resolution. A comparison of the SaMTAN-FMA complex with available Escherichia coli MTAN structures shows strong conservation of the overall structure and in particular of the active site. The presence of an extra water molecule, which forms a hydrogen bond to the O4' atom of formycin A in the active site of SaMTAN, produces electron withdrawal from the ribosyl group and may explain the lower catalytic efficiency that SaMTAN exhibits when metabolizing MTA and SAH relative to the E. coli enzyme. The implications of this structure for broad-based antibiotic design are discussed.

Class I alpha-mannosidases (glycoside hydrolase family GH47) play key roles in the maturation of N-glycans and the ER-associated degradation of unfolded glycoproteins. The 1.95 A resolution structure of a fungal alpha-1,2-mannosidase in complex with the substrate analogue methyl-alpha-D-lyxopyranosyl-(1',2)-alpha-D-mannopyranoside (LM) shows the intact disaccharide spanning the -1/+1 subsites, with the D-lyxoside ring in the -1 subsite in the 1C4 chair conformation, and provides insight into the mechanism of catalysis. The absence of the C5' hydroxymethyl group on the D-lyxoside moiety results in the side chain of Arg407 adopting two alternative conformations: the minor one interacting with Asp375 and the major one interacting with both the D-lyxoside and the catalytic base Glu409, thus disrupting its function. Chemical modification of Asp375 has previously been shown to inactivate the enzyme. Taken together, the data suggest that Arg407, which belongs to the conserved sequence motif RPExxE, may act to modulate the activity of the enzyme. The proposed mechanism for modulating the activity is potentially a general mechanism for this superfamily.

A general method for selenium and sulfur substructure determination using the Shake-and-Bake (SnB) algorithm as implemented in SnB in conjunction with anomalous difference E magnitudes is presented. The protocol can be used for Se-Met multiwavelength anomalous diffraction, seleneomethionine single anomalous diffraction (SAD), S-SAD and S/Se-single isomorphous replacement with anomalous scattering data and with minor modifications for other heavy atom derivatives, such as the location of halides.

Pyridoxal 5'-phosphate (PLP) dependent tryptophanase has been isolated from Escherichia coli and its crystal structure has been determined. The structure shares the same fold with and has similar quaternary structure to Proteus vulgaris tryptophanase and tyrosine-phenol lyase, but is found in a closed conformation when compared with these two enzymes. The tryptophanase structure, solved in its apo form, does not have covalent PLP bound in the active site, but two sulfate ions. The sulfate ions occupy the phosphoryl-binding site of PLP and the binding site of the alpha-carboxyl of the natural substrate tryptophan. One of the sulfate ions makes extensive interactions with both the transferase and PLP-binding domains of the protein and appears to be responsible for holding the enzyme in its closed conformation. Based on the sulfate density and the structure of the P. vulgaris enzyme, PLP and the substrate tryptophan were modeled into the active site. The resulting model is consistent with the roles of Arg419 in orienting the substrate to PLP and acidifying the alpha-proton of the substrate for beta-elimination, Lys269 in the formation and decomposition of the PLP quinonoid intermediate, Arg230 in orienting the substrate-PLP intermediates in the optimal conformation for catalysis, and His463 and Tyr74 in determining substrate specificity and suggests that the closed conformation observed in the structure could be induced by substrate binding and that significant conformational changes occur during catalysis. A catalytic mechanism for tryptophanase is proposed. Since E. coli tryptophanase has resisted forming diffraction-quality crystals for many years, the molecular surface of tryptophanase has been analyzed in various crystal forms and it was rationalized that strong crystal contacts occur on the flat surface of the protein and that the size of crystal contact surface seems to correlate with the diffraction quality of the crystal.

MTA/AdoHcy nucleosidase (MTAN) irreversibly hydrolyzes the N9-C1' bond in the nucleosides, 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (AdoHcy) to form adenine and the corresponding thioribose. MTAN plays a vital role in metabolic pathways involving methionine recycling, biological methylation, polyamine biosynthesis, and quorum sensing. Crystal structures of a wild-type (WT) MTAN complexed with glycerol, and mutant-enzyme and mutant-product complexes have been determined at 2.0A, 2.0A, and 2.1A resolution, respectively. The WT MTAN-glycerol structure provides a purine-free model and in combination with the previously solved thioribose-free MTAN-ADE structure, we now have separate apo structures for both MTAN binding subsites. The purine and thioribose-free states reveal an extensive enzyme-immobilized water network in their respective binding subsites. The Asp197Asn MTAN-MTA and Glu12Gln MTAN-MTR.ADE structures are the first enzyme-substrate and enzyme-product complexes reported for MTAN, respectively. These structures provide representative snapshots along the reaction coordinate and allow insight into the conformational changes of the enzyme and the nucleoside substrate. A "catalytic movie" detailing substrate binding, catalysis, and product release is presented.

An optimized technique for calculating the excess chemical potential of small molecules in dense liquids and the binding affinity of molecular ligands to biomolecules is reported. In this method, a molecular species is coupled to the system of interest via a nonphysical fourth spatial dimension w through which insertion or extraction can be carried out [R. Pomes, E. Eisenmesser, C. B. Post et al., J. Chem. Phys. 111, 3387 (1999)]. Molecular simulations are used to compute the potential of mean force (PMF) acting on the solute molecule in the fourth dimension. The excess chemical potential of that molecule is obtained as the difference in the PMF between fully coupled and fully decoupled systems. The simplicity, efficiency, and generality of the method are demonstrated for the calculation of the hydration free energies of water and methanol as well as sodium, cesium, and chloride ions. A significant advantage over other methods is that the 4D-PMF approach provides a single effective and general route for decoupling all nonbonded interactions (i.e., both Lennard-Jones and Coulombic) at once for both neutral and charged solutes. Direct calculation of the mean force from thermodynamic integration is shown to be more computationally efficient than calculating the PMF from umbrella sampling. Statistical error analysis suggests a simple strategy for optimizing sampling. The detailed analysis of systematic errors arising from the truncation of Coulombic interactions in a solvent droplet of finite size leads to straightforward corrections to ionic hydration free energies.

Delta-crystallin, the major soluble protein component in the avian eye lens, is homologous to argininosuccinate lyase (ASL). Two delta-crystallin isoforms exist in ducks, delta1- and delta2-crystallin, which are 94% identical in amino acid sequence. While duck delta2-crystallin (ddeltac2) has maintained ASL activity, evolution has rendered duck delta1-crystallin (ddeltac1) enzymatically inactive. Previous attempts to regenerate ASL activity in ddeltac1 by mutating the residues in the 20s (residues 22-31) and 70s (residues 74-89) loops to those found in ddeltac2 resulted in a double loop mutant (DLM) which was enzymatically inactive (Tsai, M. et al. (2004) Biochemistry 43, 11672-82). This result suggested that one or more of the remaining five amino acid substitutions in domain 1 of the DLM contributes to the loss of ASL activity in ddeltac1. In the current study, residues Met-9, Val-14, Ala-41, Ile-43, and Glu-115 were targeted for mutagenesis, either alone or in combination, to the residues found in ddeltac2. ASL activity was recovered in the DLM by changing Met-9 to Trp, and this activity is further potentiated in the DLM-M9W mutant when Glu-115 is changed to Asp. The roles of Trp-9 and Asp-115 were further investigated by site-directed mutagenesis in wild-type ddeltac2. Changing the identity of either Trp-9 or Asp-115 in ddeltac2 resulted in a dramatic drop in enzymatic activity. The loss of activity in Trp-9 mutants indicates a preference for an aromatic residue at this position. Truncation mutants of ddeltac2 in which the first 8, 9, or 14 N-terminal residues were removed displayed either decreased or no ASL activity, suggesting residues 1-14 are crucial for enzymatic activity in ddeltac2. Our kinetic studies combined with available structural data suggest that the N-terminal arm in ASL/delta2-crystallin is involved in stabilizing regions of the protein involved in substrate binding and catalysis, and in completely sequestering the substrate from the solvent.

Delta crystallin, a taxon-specific crystallin present in avian eye lenses, is homologous to the urea cycle enzyme ASL (argininosuccinate lyase). Although there are two delta crystallin isoforms in duck lenses, ddeltac1 (duck delta1 crystallin) and ddeltac2 (duck delta2 crystallin), only ddeltac2 is catalytically active. Previous structural studies have suggested that residues Ser283 and His162 in the multi-subunit active site of ddeltac2/ASL are the putative catalytic acid/base, while the highly conserved, positively charged Lys289 is thought to help stabilize the carbanion intermediate. The strict conservation of a small hydroxy-containing residue (Thr or Ser) at position 161 adjacent to the putative catalytic base, as well as its proximity to the substrate in the S283A ddeltac2 enzyme-substrate complex, prompted us to investigate further the role this residue. Structures of the active T161S and inactive T161D ddeltac2 mutants, as well as T161D complexed with argininosuccinate, have been determined to 2.0 A resolution. The structures suggest that a hydroxy group is required at position 161 to help correctly position the side chain of Lys289 and the fumarate moiety of the substrate. Threonine is probably favoured over serine, because the interaction of its methyl group with Leu206 would restrict its conformational flexibility. Residues larger than Thr or Ser interfere with substrate binding, supporting previous suggestions that correct positioning of the substrate's fumarate moiety is essential for catalysis to occur. The presence of the 280s loop (i.e. a loop formed by residues 270-290) in the 'open' conformation suggests that loop closure, thought to be essential for sequestration of the substrate, may be triggered by the formation of the carbanion or aci-carboxylate intermediates, whose charge distribution more closely mimics that of the sulphate ion found in the active-site region of the inactive ddeltac1. The 280s loop in ddeltac1 is in the closed conformation.

Delta-crystallin is directly related to argininosuccinate lyase (ASL), and catalyzes the reversible hydrolysis of argininosuccinate to arginine and fumarate. Two delta-crystallin isoforms exist in duck lenses, delta1 and delta2, which are 94% identical in amino acid sequence. Although the sequences of duck delta2-crystallin (ddeltac2) and duck delta1-crystallin (ddeltac1) are 69 and 71% identical to that of human ASL, respectively, only ddeltac2 has maintained ASL activity. Domain exchange experiments and comparisons of various delta-crystallin structures have suggested that the amino acid substitutions in the 20's (residues 22-31) and 70's (residues 74-89) loops of ddeltac1 are responsible for the loss of enzyme activity in this isoform. To test this hypothesis, a double loop mutant (DLM) of ddeltac1 was constructed in which all the residues that differ between the two isoforms in the 20's and 70's loops were mutated to those of ddeltac2. Contrary to expectations, kinetic analysis of the DLM found that it was enzymatically inactive. Furthermore, binding of argininosuccinate by the DLM, as well as the ddeltac1, could not be detected by isothermal titration calorimetry (ITC). To examine the conformation of the 20's and 70's loops in the DLM, and to understand why the DLM is unable to bind the substrate, its structure was determined to 2.5 A resolution. Comparison of this structure with both wild-type ddeltac1 and ddeltac2 structures reveals that the conformations of the 20's and 70's loops in the DLM mutant are very similar to those of ddeltac2. This suggests that the five amino acid substitutions in domain 1 which lie outside of the two loop regions and which are different in the DLM, and ddeltac2, must be important enzymatically. The structure of the DLM in complex with sulfate was also determined to 2.2 A resolution. This structure demonstrates that the conformational changes of the 280's loop and domain 3, previously observed in ddeltac1, also occur in the DLM upon sulfate binding, reinforcing the hypothesis that these events may occur in the active ddeltac2 protein during catalysis.

5'-Methylthioadenosine/S-adenosylhomocysteine (MTA/AdoHcy) nucleosidase is a key enzyme in a number of critical biological processes in many microbes. This nucleosidase catalyzes the irreversible hydrolysis of the N(9)-C(1') bond of MTA or AdoHcy to form adenine and the corresponding thioribose. The key role of the MTA/AdoHcy nucleosidase in biological methylation, polyamine biosynthesis, methionine recycling, and bacterial quorum sensing has made it an important antimicrobial drug target. The crystal structures of Escherichia coli MTA/AdoHcy nucleosidase complexed with the transition state analog, formycin A (FMA), and the nonhydrolyzable substrate analog, 5'-methylthiotubercidin (MTT) have been solved to 2.2- and 2.0-A resolution, respectively. These are the first MTA/AdoHcy nucleosidase structures to be solved in the presence of inhibitors. These structures clearly identify the residues involved in substrate binding and catalysis in the active site. Comparisons of the inhibitor complexes to the adenine-bound MTA/AdoHcy nucleosidase (Lee, J. E., Cornell, K. A., Riscoe, M. K., and Howell, P. L. (2001) Structure (Camb.) 9, 941-953) structure provide evidence for a ligand-induced conformational change in the active site and the substrate preference of the enzyme. The enzymatic mechanism has been re-examined.

S-Adenosylhomocysteine hydrolase (AdoHcy hydrolase) crystallizes from solutions containing the intermediate analogue neplanocin A with the analogue bound in its 3'-keto form at the active sites of all of its four subunits and the four tightly bound cofactors in their reduced (NADH) state. The enzyme is in the closed conformation, which corresponds to the structure in which the catalytic chemistry occurs. Examination of the structure in the light of available, very detailed kinetic studies [Porter, D. J., Boyd, F. L. (1991) J. Biol. Chem. 266, 21616-21625. Porter, D. J., Boyd, F. L. (1992) J. Biol. Chem. 267, 3205-3213. Porter, D. J. (1998) J. Biol. Chem. 268, 66-73] suggests elements of the catalytic strategy of AdoHcy hydrolase for acceleration of the reversible conversion of AdoHcy to adenosine (Ado) and homocysteine (Hcy). The enzyme, each subunit of which possesses a substrate-binding domain that in the absence of substrate is in rapid motion relative to the tetrameric core of the enzyme, first binds substrate and ceases motion. Probably concurrently with oxidation of the substrate to its 3'-keto form, the closed active site is "sealed off" from the environment, as indicated by a large (10(8)(-)(9)-fold) reduction in the rate of departure of ligands, a feature that prevents exposure of the labile 3'-keto intermediates to the aqueous environment. Elimination of the 5'-substituent (Hcy in the hydrolytic direction, water in the synthetic direction) generates the central intermediate 4',5'-didehydro-5'-deoxy-3'-ketoadenosine. Abortive 3'-reduction of the central intermediate is prevented by a temporary suspension of all or part of the redox catalytic power of the enzyme during the existence of the central intermediate. The abortive reduction is 10(4)-fold slower than the productive reductions at the ends of the catalytic cycle and has a rate constant similar to those of nonenzymic intramolecular model reactions. The mechanism for suspending the redox catalytic power appears to be a conformationally induced increase in the distance across which hydride transfer must occur between cofactor and substrate, the responsible conformational change again being that which "seals" the active site. The crystal structure reveals a well-defined chain of three water molecules leading from the active site to the subunit surface, which may serve as a relay for proton exchange between solvent and active site in the closed form of the enzyme, permitting maintenance of active-site functional groups in catalytically suitable protonation states.

The major soluble avian eye lens protein, delta crystallin, is highly homologous to the housekeeping enzyme argininosuccinate lyase (ASL). ASL is part of the urea and arginine-citrulline cycles and catalyzes the reversible breakdown of argininosuccinate to arginine and fumarate. In duck lenses, there are two delta crystallin isoforms that are 94% identical in amino acid sequence. Only the delta2 isoform has maintained ASL activity and has been used to investigate the enzymatic mechanism of ASL. The role of the active site residues Ser-29, Asp-33, Asp-89, Asn-116, Thr-161, His-162, Arg-238, Thr-281, Ser-283, Asn-291, Asp-293, Glu-296, Lys-325, Asp-330, and Lys-331 have been investigated by site-directed mutagenesis, and the structure of the inactive duck delta2 crystallin (ddeltac2) mutant S283A with bound argininosuccinate was determined at 1.96 A resolution. The S283A mutation does not interfere with substrate binding, because the 280's loop (residues 270-290) is in the open conformation and Ala-283 is more than 7 A from the substrate. The substrate is bound in a different conformation to that observed previously indicating a large degree of conformational flexibility in the fumarate moiety when the 280's loop is in the open conformation. The structure of the S283A ddeltac2 mutant and mutagenesis results reveal that a complex network of interactions of both protein residues and water molecules are involved in substrate binding and specificity. Small changes even to residues not involved directly in anchoring the argininosuccinate have a significant effect on catalysis. The results suggest that either His-162 or Thr-161 are responsible for proton abstraction and reinforce the putative role of Ser-283 as the catalytic acid, although we cannot eliminate the possibility that arginine is released in an uncharged form, with the solvent providing the required proton. A detailed enzymatic mechanism of ASL/ddeltac2 is presented.