Catalytic His Research Articles

Exploring the pH dependence of an improved PETase

Enzymatic recycling of plastic and especially of polyethylene terephthalate (PET) has shown great potential to reduce its negative impact on our society. PET hydrolases (PETases) have been optimized using rational design and machine learning, but the mechanistic details of the PET depolymerization process remain unclear. Belonging to the carboxylic-ester hydrolase family with a canonical Ser-His-Asp catalytic triad, their observed alkaline pH optimum is generally thought to be related to the protonation state of the catalytic His. Here, we explore this aspect in the context of LCCICCG, an optimized PETase, derived from the leaf-branch compost cutinase enzyme. We use NMR to identify the dominant tautomeric structure of the six histidines. Five show surprisingly low pKa values below 4.0, whereas the catalytic H242 in the active enzyme displays a pKa value that varies from 4.9 to 4.7 when temperatures increase from 30°C to 50°C. Whereas the hydrolytic activity of the enzyme toward a soluble substrate can be modeled by the corresponding protonation/deprotonation curve, an important discrepancy is found when the substrate is the solid plastic. This opens the way to further mechanistic understanding of the PETase activity and underscores the importance of studying the enzyme at the liquid-solid interface.

Identification and Characterization of a Novel Thermostable GDSL Lipase LipGt6 from Geobacillus thermoleovorans H9

Lipases are versatile biocatalysts for various biological reactions. In the detergent industry, lipases must exhibit high activity in environments with high temperature, high pH values, metal ions, and organic solvents. Therefore, researchers are intensively searching for more stable and efficient lipases. A new thermophilic lipase, LipGt6, was identified in Geobacillus thermoleovorans H9, a new thermophilic strain isolated from ultrahigh-temperature compost. A structural model of LipGt6 was constructed using an esterase from Geobacillus thermodenitrificans as a template, and site-directed mutagenesis confirmed the predicted active site residues. LipGt6 exhibited the highest activity towards medium- and long-chain fatty acids (C8–C14), and the optimum temperature and pH were 50 °C and 9.0, respectively. LipGt6 was found to be thermostable up to 70 °C. In the presence of 1% H2O2 and sodium deoxycholate, LipGt6 retained 70 to 75% relative activity. These findings reveal that LipGt6 is potentially useful for the industrial production of detergent. Based on comparison of the amino acid sequences, the enzyme belongs to a new subfamily called lipolytic enzyme family II. The catalytic residues Ser and His were more critical than Asp, and the Asp221 catalytic residue is not likely critical for the lipolytic reaction of LipGt6.

Revisiting catalytic His and Glu residues in coproporphyrin ferrochelatase - unexpected activities of active site variants.

The identification of the coproporphyrin-dependent heme biosynthetic pathway, which is used almost exclusively by monoderm bacteria in 2015 by Dailey etal. triggered studies aimed at investigating the enzymes involved in this pathway that were originally assigned to the protoporphyrin-dependent heme biosynthetic pathway. Here, we revisit the active site of coproporphyrin ferrochelatase by a biophysical and biochemical investigation using the physiological substrate coproporphyrin III, which in contrast to the previously used substrate protoporphyrin IX has four propionate substituents and no vinyl groups. In particular, we have compared the reactivity of wild-type coproporphyrin ferrochelatase from the firmicute Listeria monocytogenes with those of variants, namely, His182Ala (H182A) and Glu263Gln (E263Q), involving two key active site residues. Interestingly, both variants are active only toward the physiological substrate coproporphyrin III but inactive toward protoporphyrin IX. In addition, E263 exchange impairs the final oxidation step from ferrous coproheme to ferric coproheme. The characteristics of the active site in the context of the residues involved and the substrate binding properties are discussed here using structural and functional means, providing a further contribution to the deciphering of this enigmatic reaction mechanism.

The 1.7 Å crystal structure of the C5a peptidase from Streptococcus agalactiae (ScpB) reveals an active site competent for catalysis.

A 1.7 Å structure is presented for an active form of the virulence factor ScpB, the C5a peptidase from Streptococcus agalactiae. The previously reported structure of the ScpB active site mutant exhibited a large separation (~20 Å) between the catalytic His and Ser residues. Significant differences are observed in the catalytic domain between the current and mutant ScpB structures resulting with a high RMSDCα (4.6 Å). The fold of the active form of ScpB is nearly identical to ScpA (RMSDCα 0.2 Å), the C5a-peptidase from Streptococcus pyogenes. Both ScpA and ScpB have comparable activity against human C5a, indicating neither enzyme require host proteins for C5a-ase activity. These studies are a first step in resolving reported differences in the specificities of these enzymes.

X-ray structure of recombinant house dust mite allergen Der p 3

The X-ray structure of the Der p 3 allergen was solved at 2.25 Å resolution using the molecular replacement method. The most remarkable difference in conformation of the polypeptide chains between the Der p 3 allergen and bovine trypsin is observed in the disordered parts of the polypeptide chain where the degree of homology is lower. The active site of the Der p 3 allergen which involves the residues of catalytic triad Ser197, His 52, and Asp97 (Ser195, His57, and Asp102 in bovine trypsin) responsible for the specific binding of the positively charged substrates Asp191 (Asp189 in bovine trypsin) is in the cleft between the domains.

Functional and structural properties of pyridoxal reductase (PdxI) from Escherichia coli: a pivotal enzyme in the vitamin B6 salvage pathway.

Pyridoxine 4-dehydrogenase (PdxI), a NADPH-dependent pyridoxal reductase, is one of the key players in the Escherichia coli pyridoxal 5'-phosphate (PLP) salvage pathway. This enzyme, which catalyses the reduction of pyridoxal into pyridoxine, causes pyridoxal to be converted into PLP via the formation of pyridoxine and pyridoxine phosphate. The structural and functional properties of PdxI were hitherto unknown, preventing a rational explanation of how and why this longer, detoured pathway occurs, given that, in E. coli, two pyridoxal kinases (PdxK and PdxY) exist that could convert pyridoxal directly into PLP. Here, we report a detailed characterisation of E. coli PdxI that explains this behaviour. The enzyme efficiently catalyses the reversible transformation of pyridoxal into pyridoxine, although the reduction direction is thermodynamically strongly favoured, following a compulsory-order ternary-complex mechanism. In vitro, the enzyme is also able to catalyse PLP reduction and use NADH as an electron donor, although with lower efficiency. As with all members of the aldo-keto reductase (AKR) superfamily, the enzyme has a TIM barrel fold; however, it shows some specific features, the most important of which is the presence of an Arg residue that replaces the catalytic tetrad His residue that is present in all AKRs and appears to be involved in substrate specificity. The above results, in conjunction with kinetic and static measurements of vitamins B6 in cell extracts of E. coli wild-type and knockout strains, shed light on the role of PdxI and both kinases in determining the pathway followed by pyridoxal in its conversion to PLP, which has a precise regulatory function.

C-Terminal β8-α9 Interaction Modulates Thermal Stability and Enzymatic Activity Differently in Hyperthermophilic Esterase EstE1 and Mesophilic Esterase rPPE.

Hydrophobic interactions and hydrogen bonds are 2 types of noncovalent interactions that play distinct roles in the folding and structural stability of proteins. However, the specific roles of these interactions in hydrophobic or hydrophilic environments in α/β-hydrolases are not fully understood. A hyperthermophilic esterase EstE1 in a dimer maintains the C-terminal β8-α9 strand-helix via hydrophobic interactions (Phe276 and Leu299), constituting a closed dimer interface. Moreover, a mesophilic esterase rPPE in a monomer maintains the same strand-helix via a hydrogen bond (Tyr281 and Gln306). Unpaired polar residues (F276Y in EstE1 and Y281A/F and Q306A in rPPE) or reduced hydrophobic interactions (F276A/L299A in EstE1) between the β8-α9 strand-helix decrease thermal stability. EstE1 (F276Y/L299Q) and rPPE WT, both with the β8-α9 hydrogen bond, showed the same thermal stability as EstE1 WT and rPPE (Y281F/Q306L), which possess hydrophobic interactions instead. However, EstE1 (F276Y/L299Q) and rPPE WT exhibited higher enzymatic activity than EstE1 WT and rPPE (Y281F/Q306L), respectively. This suggests that α/β-hydrolases favor the β8-α9 hydrogen bond for catalytic activity in monomers or oligomers. Overall, these findings demonstrate how α/β-hydrolases modulate hydrophobic interactions and hydrogen bonds to adapt to different environments. Both types of interactions contribute equally to thermal stability, but the hydrogen bond is preferred for catalytic activity. IMPORTANCE Esterases hydrolyze short to medium-chain monoesters and contain a catalytic His on a loop between the C-terminal β8-strand and α9-helix. This study explores how hyperthermophilic esterase EstE1 and mesophilic esterase rPPE adapt to different temperatures by utilizing the β8-α9 hydrogen bonds or hydrophobic interactions differently. EstE1 forms a hydrophobic dimer interface, while rPPE forms a monomer stabilized by a hydrogen bond. The study demonstrates that these enzymes stabilize β8-α9 strand-helix differently but achieve similar thermal stability. While the β8-α9 hydrogen bond or hydrophobic interactions contribute equally to thermal stability, the hydrogen bond provides higher activity due to increased catalytic His loop flexibility in both EstE1 and rPPE. These findings reveal how enzymes adapt to extreme environments while maintaining their functions and have implications for engineering enzymes with desired activities and stabilities.

Action mechanism of snake venom l-amino acid oxidase and its double-edged sword effect on cancer treatment: Role of pannexin 1-mediated interleukin-6 expression

Snake venom l-amino acid oxidases (svLAAOs) have been recognized as promising candidates for anticancer therapeutics. However, multiple aspects of their catalytic mechanism and the overall responses of cancer cells to these redox enzymes remain ambiguous. Here, we present an analysis of the phylogenetic relationships and active site-related residues among svLAAOs and reveal that the previously proposed critical catalytic residue His 223 is highly conserved in the viperid but not the elapid svLAAO clade. To gain further insight into the action mechanism of the elapid svLAAOs, we purify and characterize the structural, biochemical, and anticancer therapeutic potentials of the Thailand elapid snake Naja kaouthia LAAO (NK-LAAO). We find that NK-LAAO, with Ser 223, exhibits high catalytic activity toward hydrophobic l-amino acid substrates. Moreover, NK-LAAO induces substantial oxidative stress-mediated cytotoxicity with the magnitude relying on both the levels of extracellular hydrogen peroxide (H2O2) and intracellular reactive oxygen species (ROS) generated during the enzymatic redox reactions, but not being influenced by the N-linked glycans on its surface. Unexpectedly, we discover a tolerant mechanism deployed by cancer cells to dampen the anticancer activities of NK-LAAO. NK-LAAO treatment amplifies interleukin (IL)-6 expression via the pannexin 1 (Panx1)-directed intracellular calcium (iCa2+) signaling pathway to confer adaptive and aggressive phenotypes on cancer cells. Accordingly, IL-6 silencing renders cancer cells vulnerable to NK-LAAO-induced oxidative stress together with abrogating NK-LAAO-stimulated metastatic acquisition. Collectively, our study urges caution when using svLAAOs in cancer treatment and identifies the Panx1/iCa2+/IL-6 axis as a therapeutic target for improving the effectiveness of svLAAOs-based anticancer therapies.

N-acetylglucosamine-phosphatidylinositol de-N-acetylase as a novel target for probing potential inhibitor against Leishmania donovani

Leishmania donavani is the causative agent of leishmaniasis, responsible for social and economic disruption, especially in developing countries. Lack of effective drugs with few side effects have necessitated the discovery of newer therapeutic solutions for leishmaniasis. Glycophosphatidylinositol (GPI) synthesis plays a vital role in protozoan cell membranes structural formation and antigenic modification. Hence, any disruption in its biosynthesis can prove fatal to the parasitic protozoans. N-acetylglucosamine-phosphatidylinositol de-N-acetylase (NAGP-deacetylase) is an enzyme from the GPI biosynthetic pathway that catalyzes the deacetylation of N-acetylglucosaminylphosphatidylinositol to glucosaminylphosphatidylinositol, a step essential for the proper functioning of the enzyme. In the quest for novel scaffolds as anti-leishmaniasis agents, we have executed in silico virtual screening, density function theory, molecular dynamics and MM-GBSA based energy calculations with a natural product library and a diverse library set from Chembridge database. Two compounds, 14671 and 4610, were identified at the enzyme's active site and interacted with catalytic residues, Asp43, Asp44, His41, His147, His 150, Arg80 and Arg231. Both molecules exhibited stable conformation in their protein-ligand complexes with binding free energies for compound-14671 and compound-4610 of −54 ± 4 and −50 ± 4 kcal/mol, respectively. These scaffolds can be incorporated in future synthetic determinations, focusing on developing druggable inhibitor support, increasing potency, and introducing species selectivity. Communicated by Ramaswamy H. Sarma

The Crystal Structure of Nα-p-tosyl-lysyl Chloromethylketone-Bound Oligopeptidase B from Serratia Proteamaculans Revealed a New Type of Inhibitor Binding

A covalent serine protease inhibitor—Na-p-Tosyl-Lysyl Chloromethylketone (TCK) is a modified lysine residue tosylated at the N-terminus and chloromethylated at the C-terminus, one molecule of which is capable of forming two covalent bonds with both Ser and His catalytic residues, was co-crystallized with modified oligopeptidase B (OpB) from Serratia proteomaculans (PSPmod). The kinetics study, which preceded crystallization, shows that the stoichiometry of TCK-dependent inhibition of PSPmod was 1:2 (protein:inhibitor). The crystal structure of the PSPmod-TCK complex, solved at a resolution of 2.3 Å, confirmed a new type of inhibitor binding. Two TCK molecules were bound to one enzyme molecule: one with the catalytic Ser, the other with the catalytic His. Due to this mode of binding, the intermediate state of PSPmod and the disturbed conformation of the catalytic triad were preserved in the PSPmod-TCK complex. Nevertheless, the analysis of the amino acid surroundings of the inhibitor molecule bound to the catalytic Ser and its comparison with that of antipain-bound OpB from Trypanosoma brucei provided an insight in the structure of the PSPmod substrate-binding pocket. Supposedly, the new type of binding is typical for the interaction of chloromethylketone derivatives with two-domain OpBs. In the open conformational state that these enzymes are assumed in solution, the disordered configuration of the catalytic triad prevents simultaneous interaction of one inhibitor molecule with two catalytic residues.

Role of endopeptidases in peptidoglycan synthesis mediated by alternative cross-linking enzymes in Escherichia coli.

Bacteria resist to the turgor pressure of the cytoplasm through a net-like macromolecule, the peptidoglycan, made of glycan strands connected via peptides cross-linked by penicillin-binding proteins (PBPs). We recently reported the emergence of β-lactam resistance resulting from a bypass of PBPs by the YcbB L,D-transpeptidase (LdtD), which form chemically distinct 3→3 cross-links compared to 4→3 formed by PBPs. Here we show that peptidoglycan expansion requires controlled hydrolysis of cross-links and identify among eight endopeptidase paralogues the minimum enzyme complements essential for bacterial growth with 4→3 (MepM) and 3→3 (MepM and MepK) cross-links. Purified Mep endopeptidases unexpectedly displayed a 4→3 and 3→3 dual specificity implying recognition of a common motif in the two cross-link types. Uncoupling of the polymerization of glycan chains from the 4→3 cross-linking reaction was found to facilitate the bypass of PBPs by YcbB. These results illustrate the plasticity of the peptidoglycan polymerization machinery in response to the selective pressure of β-lactams.

The Role of Conserved Residues in the DEDDh Motif: the Proton-Transfer Mechanism of HIV-1 RNase H

RNase H is a prototypical example for two-metal-ion catalysis in enzymes. An RNase H activity cleaving the ribonucleic acid (RNA) backbone of a DNA/RNA hybrid is present not only in important drug targets, such as the HIV-1 reverse transcriptase, but also in many other nucleases, such as Homo sapiens (Hs) and Escherichia coli (Ec) RNase H or, notably, in enzymes that are part of the CRISPR gene editing molecular machinery. Despite its importance, the reaction mechanism uncovering the proton-transfer events is not yet understood. In particular, it is not known, which group is the proton donor for the leaving group. Moreover, several different proton acceptors were proposed, and the exact identity of the proton acceptor is also elusive. Here, we revisit the mechanism for RNAse H, whereby we find that the highly conserved Glu residue of the DDE motif acts as a proton donor via a mechanism further stabilized by the 2′O atom of the sugar. Additionally, we also describe an alternative proton-transfer mechanism via a conserved catalytic His residue to deprotonate the attacking water molecule. Furthermore, our quantum mechanics/molecular mechanics (QM/MM) calculations combining Hamiltonian replica exchange with a finite-temperature string method provide an accurate free-energy profile for the reaction catalyzed by the HIV-1 RNase H. Our reported pathway is consistent with kinetic data obtained for mutant HIV-1, Hs, and Ec RNase H, with the calculated pKa values of the DEDD residues and with crystallographic studies. The overall reaction barrier of ∼19 kcal mol–1, encountered in the phosphate-cleavage step, matches the slow experimental rate of ∼1–100 min–1. Additionally, using molecular dynamics (MD) calculations, we sample the recently identified binding site for a third transient divalent metal ion in the vicinity of the scissile phosphate in the product complex. Our results account for the experimental observation of a third metal ion facilitating product release in an Aquifex aeolicus RNase III crystal structure and the Bh RNase H in crystallo reaction. Taken together, we provide a molecular mechanism of the nuclease catalytic reaction that is likely common for the broad family of two-metal-ion catalytic phosphate-cleaving enzymes with a DDE motif.

Catalytically active peptides affected by self-assembly and residues order

Amphiphilic peptides that induce catalysis are interesting alternatives to natural enzymes thanks to robustness of their synthesis and the ability to induce certain types of conformations by specific motifs of amino acid sequences. Various studies aimed at mimicking the activity of serine proteases by designed peptides. Here we demonstrate that the order by which the catalytic triad residues are positioned along amphiphilic β-strands influences both assembly structures and catalytic activity. A set of three β-sheet amphiphilic peptides, decorated with different orders of the catalytic triad amino acids, Glu, His and Ser along the strands were evaluated for their catalytic hydrolysis efficiency of p-nitrophenyl acetate (pNPA) substrate. Among the three peptides, Ac-Cys-Phe-Glu-Phe-Ser-Phe-His-Phe-Pro-NH2 (ESH) achieved the greatest catalytic efficiency with a value of 0.19 M−1 s−1, at peptide concentration of 250 μM. This study sheds light on an overlooked factor in designing catalytic amphiphilic assemblies whereby charged residues that make up the active sites, are in fact engaged in intermolecular stabilizing interactions that in turn may hamper their catalytic action.

Revisiting the pH-gated conformational switch on the activities of HisKA-family histidine kinases

Histidine is a versatile residue playing key roles in enzyme catalysis thanks to the chemistry of its imidazole group that can serve as nucleophile, general acid or base depending on its protonation state. In bacteria, signal transduction relies on two-component systems (TCS) which comprise a sensor histidine kinase (HK) containing a phosphorylatable catalytic His with phosphotransfer and phosphatase activities over an effector response regulator. Recently, a pH-gated model has been postulated to regulate the phosphatase activity of HisKA HKs based on the pH-dependent rotamer switch of the phosphorylatable His. Here, we have revisited this model from a structural and functional perspective on HK853–RR468 and EnvZ–OmpR TCS, the prototypical HisKA HKs. We have found that the rotamer of His is not influenced by the environmental pH, ruling out a pH-gated model and confirming that the chemistry of the His is responsible for the decrease in the phosphatase activity at acidic pH.

Identification of potential SARS-CoV-2 main protease inhibitors from Ficus Carica Latex: An in-silico approach

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is the aetiological agent behind the current pandemic of coronavirus disease 2019 (COVID-19). SARS-CoV-2 main protease plays a dynamic role in mediating viral replication and transcription, which is one of the most probable drug targets against SARS-CoV-2. Ficus carica latex encompasses notable bioactive molecules with various biological properties, including antiviral activities. In this study, latex compounds of Ficus carica were screened to find out active phytochemicals against SARS-CoV-2 main protease through molecular docking, molecular dynamics simulation, and ADMET (absorption, distribution, metabolism, excretion, and toxicity) profiling. A total of 21 compounds were screened, and the compounds, lupeol, α-amyrin, and luteolin, showed the highest binding affinity and intense interaction with the vital catalytic residue His 41 and Cys 145. The molecular dynamics simulation revealed that the amyrin is the most stable compound with higher binding free energy, suggesting that this compound can compete with the native ligands of the main protease. The ADMET analysis indicated that these phytochemicals have considerable physicochemical, pharmacokinetics, and drug-likeness properties and do not possess any considerable detrimental effects and can be considered as potential drug candidates against SARS-CoV-2. However, further in-vitro, in-vivo, and clinical trials are required to observe the exact efficiency of these compounds. © 2020, Bangladesh Society for Microbiology, Immunology and Advanced Biotechnology. All rights reserved.

Protein Phosphohistidine Phosphatases of the HP Superfamily.

Histidine phosphorylation of proteins is increasingly recognised as an important regulatory posttranslational modification in eukaryotes as well as prokaryotes. The HP (Histidine Phosphatase) superfamily, named for a key catalytic His residue, harbors two known groups of protein phosphohistidine phosphatases (PPHPs). The bacterial SixA protein acts as a regulator of His-Asp phosphorelays with two substrates characterized in vitro and/or in vivo. The recently characterized eukaryotic PHPP PGAM5 only has one currently known substrate, NDPK-B, through which it helps regulate T-cell signaling. SixA and PGAM5 appear to share no particular sequence or structural features relating to their PPHP activity suggesting that PHPP activity has arisen independently in different lineages of the HP superfamily. Further members of the HP superfamily may thus harbor (additional) unsuspected PHPP activity.

Sequence and Structural Analysis of AA9 and AA10 LPMOs: An Insight into the Basis of Substrate Specificity and Regioselectivity.

Lytic polysaccharide monooxygenases (LPMOs) are key enzymes in both the natural carbon cycle and the biorefinery industry. Understanding the molecular basis of LPMOs acting on polysaccharide substrates is helpful for improving industrial cellulase cocktails. Here we analyzed the sequences, structures, and substrate binding modes of LPMOs to uncover the factors that influence substrate specificity and regioselectivity. Our results showed that the different compositions of a motif located on L2 affect the electrostatic potentials of substrate binding surfaces, which in turn affect substrate specificities of AA10 LPMOs. A conserved Asn at a distance of 7 Å from the active center Cu might, together with the conserved Ser immediately before the second catalytic His, determine the localization of LPMOs on substrate, and thus contribute to C4-oxidizing regioselectivity. The findings in this work provide an insight into the molecular basis of substrate specificity and regioselectivity of LPMOs.

Expression, purification and characterisation of Trypanosoma congolense metacaspase 5 (TcoMCA5) - a potential drug target for animal African trypanosomiasis

The metacaspases (MCAs) are attractive drug targets for the treatment of African trypanosomiasis as they are not found in the metazoan kingdom and their action has been implicated in cell cycle and cell death pathways in kinetoplastid parasites. Here we report the biochemical characterisation of MCA5 from T. congolense. Upon recombinant expression in E. coli, autoprocessing is evident, and MCA5 further autoprocesses when purified using nickel affinity chromatography, which we term nickel-induced over autoprocessing. When both the catalytic His and Cys residues were mutated (TcoMCA5H147A/C202G), no nickel-induced over autoprocessing was observed and was enzymatically active, suggesting the existence of a secondary catalytic Cys residue, Cys81. Immunoaffinity purification of native TcoMCA5 from the total parasite proteins was achieved using chicken anti-TcoMCA5 IgY antibodies. The full length native TcoMCA5 and the autoprocessed products of recombinant TcoMCA5H147A/C202G were shown to possess gelatinolytic activity, the first report for that of a MCA. Both the native and recombinant enzyme were calcium independent, had a preference for Arg over Lys at the P1 site and were active over a pH range between 6.5 and 9. Partial inhibition (23%) of enzymatic activity was only achieved with leupeptin and antipain. These findings are the first step in the biochemical characterisation of the single copy MCAs from animal infective trypanosomes towards the design of novel trypanocides.

Distinct roles of an ionic interaction holding an alpha-helix with catalytic Asp and a beta-strand with catalytic His in a hyperthermophilic esterase EstE1 and a mesophilic esterase rPPE

An ionic interaction that holds an α-helix and a β-strand on which catalytic Asp and His residues are located, respectively, is conserved in a hyperthermophilic esterase EstE1 (optimum temperature 70°C) and a mesophilic esterase rPPE (optimum temperature 50°C). We investigated the role of an ionic interaction between E258 and R275 in EstE1 and that between E263 and R280 in rPPE in active-site stability of serine esterases adapted to different temperatures. Ala substitutions caused a 5-10°C decrease in the optimum temperature of both EstE1 and rPPE mutants. Surprisingly, disruption of the ionic interaction caused larger effects on the conformational flexibility of EstE1 mutants despite their rigid structures, whereas the disruption had fewer effects on the thermal stability of EstE1 mutants at 60-70°C, as the structure of EstE1 was adapted to high temperatures. In contrast, mesophilic rPPE mutants showed dramatic decreases in thermal stability at 40-50°C, but less changes in conformational flexibility because of their inherently flexible structures. The results of this study suggest that the ionic interaction between the α-helix with catalytic Asp and the β-strand with catalytic His plays an important role in the active-site conformation of EstE1 and rPPE, with larger effects on the conformational flexibility of hyperthermophilic EstE1 and the thermal stability of mesophilic rPPE.

Structural analysis of a Vibrio phospholipase reveals an unusual Ser–His–chloride catalytic triad

Phospholipases can disrupt host membranes and are important virulence factors in many pathogens. VvPlpA is a phospholipase A2 secreted by Vibrio vulnificus and essential for virulence. Its homologs, termed thermolabile hemolysins (TLHs), are widely distributed in Vibrio bacteria, but no structural information for this virulence factor class is available. Herein, we report the crystal structure of VvPlpA to 1.4-Å resolution, revealing that VvPlpA contains an N-terminal domain of unknown function and a C-terminal phospholipase domain and that these two domains are packed closely together. The phospholipase domain adopts a typical SGNH hydrolase fold, containing the four conserved catalytic residues Ser, Gly, Asn, and His. Interestingly, the structure also disclosed that the phospholipase domain accommodates a chloride ion near the catalytic His residue. The chloride is five-coordinated in a distorted bipyramid geometry, accepting hydrogen bonds from a water molecule and the amino groups of surrounding residues. This chloride substitutes for the most common Asp/Glu residue and forms an unusual Ser-His-chloride catalytic triad in VvPlpA. The chloride may orient the catalytic His and stabilize the charge on its imidazole ring during catalysis. Indeed, VvPlpA activity depended on chloride concentration, confirming the important role of chloride in catalysis. The VvPlpA structure also revealed a large hydrophobic substrate-binding pocket that is capable of accommodating a long-chain acyl group. Our results provide the first structure of the TLH family and uncover an unusual Ser-His-chloride catalytic triad, expanding our knowledge on the biological role of chloride.