Lowering pH optimum of activity of SshEstI, a slightly alkaliphilic archaeal esterase of the hormone-sensitive lipase family.

SshEstI, a carboxylesterase from the thermoacidophilic archaeon Saccharolobus shibatae, is a member of the hormone-sensitive lipase family that displays slightly alkaliphilic activity with an optimum activity at pH 8.0. In this study, three distinct strategies were explored to confer acidophilic properties to SshEstI. The first strategy involved engineering the oxyanion hole by replacing Gly81 with serine or aspartic acid. The G81S mutant showed optimum activity at pH 7.0, whereas the aspartic acid mutant (G81D) rendered the enzyme slightly acidophilic with optimum activity observed at pH 6.0; however, kcat and kcat/Km values were reduced by these substitutions. The second strategy involved examining the effects of surfactant additives on the pH-activity profiles of SshEstI. The results showed that cetyltrimethylammonium bromide (CTAB) enhanced wild-type enzyme (WT) activity at acidic pH values. In the presence of 0.1 mM CTAB, G81S and G81D were acidophilic enzymes with optimum activity at pH 6.0 and 4.0, respectively, although their enzyme activities were low. The third strategy involved engineering the active site to resemble that of kumamolisin-As (kuma-As), an acidophilic peptidase of the sedolisin family. The catalytic triad of kuma-As was exchanged into SshEstI using site-directed mutagenesis. X-ray crystallographic analysis of the mutants (H274D and H274E) revealed that the potential hydrogen donor-acceptor distances around the active site of WT were fully maintained in these mutants. However, these mutants were inactive at pH 4-8.

Carbohydrate-binding ability of a recombinant protein containing the DM9 motif from Drosophila melanogaster.

Proteins containing DM9 motifs, which were originally identified in the Drosophila melanogaster genome, are widely distributed in various organisms and are assumed to be involved in their innate immune response. In this study, we produced a recombinant protein of CG13321 (rCG13321) from D. melanogaster, which consists of four DM9 motifs, in Escherichia coli cells. In affinity chromatography using a mannose-immobilized column, rCG13321 exhibited mannose-binding ability and was separated into high-affinity and low-affinity fractions, named HA and LA, respectively, based on its binding ability to the column. In addition to having a higher affinity for the column, HA exhibited self-oligomerization ability, suggesting slight differences in tertiary structure. Both LA and HA showed hemagglutinating activity and were able to agglutinate an oligomannose-containing dendrimer, indicating that they have multiple carbohydrate-binding sites. Glycan array analysis suggested that rCG13321 primarily recognizes d-mannose and d-rhamnose through hydrogen bonding with the 2-, 3- and 4-hydroxy groups. Isothermal titration calorimetry demonstrated that rCG13321 has a comparable affinity to typical lectins. These findings suggest that CG13321 functions as a carbohydrate-binding protein or lectin that recognizes mannose and related carbohydrate-containing molecules on the surface of foreign organisms as a pattern recognition molecule.

Mannose oligosaccharide recognition of CGL1, a mannose-specific lectin containing DM9 motifs from Crassostrea gigas, revealed by X-ray crystallographic analysis.

CGL1 is a mannose-specific lectin isolated from the Pacific oyster Crassostrea gigas, and it belongs to the DM9 domain protein family. Each subunit of the CGL1 dimer consists of a tandem repeat of DM9 motifs, which were originally found in the Drosophila melanogaster genome. The CGL1 protomer contains two carbohydrate-binding sites: a high-affinity site A and a low-affinity site B. An assay using dendrimers containing oligomannose from yeast (Saccharomyces cerevisiae) revealed that CGL1 exhibited significantly higher affinity for mannotetraose (Man4) compared to mannobiose (Man2) and mannotriose (Man3). To investigate its oligomannose-recognition mechanism, X-ray crystallographic analyses of CGL1/oligomannose complexes were performed. In the CGL1/Man2 and CGL1/Man3 complexes, Manα1-2Man and Manα1-2Manα1-2Man, respectively, were primarily bound to site A, interacting with the non-reducing mannose residue. On the other hand, in the CGL1/Man4 crystal, Man4 (Manα1-2Manα1-2Manα1-6Man) was bound at both site A and site B at the non-reducing and reducing ends, thus linking adjacent CGL1 molecules with crystallographic symmetry. These findings suggest that CGL1 can recognize both the non-reducing and reducing mannose residues of mannose oligosaccharides at its two distinct carbohydrate-binding sites. This enables efficient complex formation, making CGL1 a pattern-recognition molecule capable of recognizing diverse structures of mannose-containing carbohydrate chains.

Functional Diversity of Novel Lectins with Unique Structural Features in Marine Animals.

Due to their remarkable structural diversity, glycans play important roles as recognition molecules on cell surfaces of living organisms. Carbohydrates exist in numerous isomeric forms and can adopt diverse structures through various branching patterns. Despite their relatively small molecular weights, they exhibit extensive structural diversity. On the other hand, lectins, also known as carbohydrate-binding proteins, not only recognize and bind to the diverse structures of glycans but also induce various biological reactions based on structural differences. Initially discovered as hemagglutinins in plant seeds, lectins have been found to play significant roles in cell recognition processes in higher vertebrates. However, our understanding of lectins in marine animals, particularly marine invertebrates, remains limited. Recent studies have revealed that marine animals possess novel lectins with unique structures and glycan recognition mechanisms not observed in known lectins. Of particular interest is their role as pattern recognition molecules in the innate immune system, where they recognize the glycan structures of pathogens. Furthermore, lectins serve as toxins for self-defense against foreign enemies. Recent discoveries have identified various pore-forming proteins containing lectin domains in fish venoms and skins. These proteins utilize lectin domains to bind target cells, triggering oligomerization and pore formation in the cell membrane. These findings have spurred research into the new functions of lectins and lectin domains. In this review, we present recent findings on the diverse structures and functions of lectins in marine animals.

Molecular cloning and characterization of the two putative toxins expressed in the venom of the devil stinger Inimicus japonicus.

Various proteins are involved in fish venom toxicity, but limited information is available regarding their structure and mode of action. Here, we analyzed RNA transcripts in the dorsal spine of the devil stinger Inimicus japonicus using next-generation sequencing (NGS), and identified two putative protein toxins, a natterin-like protein (Ij-natterin) and a phospholipase A2 (Ij-PLA2), as well as a previously reported stonustoxin-like protein. The deduced amino acid sequence of Ij-natterin suggested that it acts as a pore-forming toxin through the cooperation of the N-terminal lectin-like domain and the C-terminal pore-forming domain. Ij-PLA2 showed significant homology with secreted Ca2+-dependent PLA2s from snake venom and mammals (sPLA2-I/II). The recombinant Ij-PLA2 protein exhibited PLA2 activity in the absence of Ca2+, in contrast to canonical sPLA2-I/II. Comparison of the amino acid sequences of Ij-PLA2 with the other sPLA2-I/II suggests that the C-terminal extended peptide region of Ij-PLA2 is involved in its Ca2+-independent activity.

A versatile cis-prenyltransferase from Methanosarcina mazei catalyzes both C- and O-prenylations.

Polyprenyl groups, products of isoprenoid metabolism, are utilized in peptidoglycan biosynthesis, protein N-glycosylation, and other processes. These groups are formed by cis-prenyltransferases, which use allylic prenyl pyrophosphates as prenyl-donors to catalyze the C-prenylation of the general acceptor substrate, isopentenyl pyrophosphate. Repetition of this reaction forms (Z,E-mixed)-polyprenyl pyrophosphates, which are converted later into glycosyl carrier lipids, such as undecaprenyl phosphate and dolichyl phosphate. MM_0014 from the methanogenic archaeon Methanosarcina mazei is known as a versatile cis-prenyltransferase that accepts both isopentenyl pyrophosphate and dimethylallyl pyrophosphate as acceptor substrates. To learn more about this enzyme's catalytic activity, we determined the X-ray crystal structures of MM_0014 in the presence or absence of these substrates. Surprisingly, one structure revealed a complex with O-prenylglycerol, suggesting that the enzyme catalyzed the prenylation of glycerol contained in the crystallization buffer. Further analyses confirmed that the enzyme could catalyze the O-prenylation of small alcohols, such as 2-propanol, expanding our understanding of the catalytic ability of cis-prenyltransferases.

Structural analysis and reaction mechanism of the disproportionating enzyme (D-enzyme) from potato.

Starch produced by plants is a stored form of energy and is an important dietary source of calories for humans and domestic animals. Disproportionating enzyme (D-enzyme) catalyzes intramolecular and intermolecular transglycosylation reactions of α-1, 4-glucan. D-enzyme is essential in starch metabolism in the potato. We present the crystal structures of potato D-enzyme, including two different types of complex structures: a primary Michaelis complex (substrate binding mode) for 26-meric cycloamylose (CA26) and a covalent intermediate for acarbose. Our study revealed that the acarbose and CA26 reactions catalyzed by potato D-enzyme involve the formation of a covalent intermediate with the donor substrate. HPAEC of reaction substrates and products revealed the activity of the potato D-enzyme on acarbose and CA26 as donor substrates. The structural and chromatography analyses provide insight into the mechanism of the coupling reaction of CA and glucose catalyzed by the potato D-enzyme. The enzymatic reaction mechanism does not involve residual hydrolysis. This could be particularly useful in preventing unnecessary starch degradation leading to reduced crop productivity. Optimization of this mechanism would be important for improvements of starch storage and productivity in crops.

Novel carbohydrate-recognition mode of the invertebrate C-type lectin SPL-1 from Saxidomus purpuratus revealed by the GlcNAc-complex crystal in the presence of Ca2.

The C-type lectins SPL-1 and SPL-2 from the bivalve Saxidomus purpuratus are composed of A and B chains and of two B chains, respectively. They bind specific carbohydrates containing acetamido groups, such as N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc), in a Ca2+-independent manner. Unlike ordinary C-type lectins, which require Ca2+ ions for carbohydrate recognition, these lectins recognize specific carbohydrates mainly through interactions with the acetamido group without Ca2+ ions, even though Ca2+ enhances the binding affinity of these lectins, especially SPL-1. In the present study, the crystal structure of the SPL-1-GlcNAc complex in the presence of Ca2+ revealed that the binding of SPL-1 to GlcNAc is stabilized by hydrogen bonds to the water molecule(s) coordinating Ca2+, whereas in ordinary C-type lectins Ca2+ directly forms coordinate bonds to the hydroxy groups of carbohydrates. These differences may also allow SPL-1 and SPL-2 to recognize both GlcNAc and GalNAc, which have different orientations of the 4-hydroxy group.

Mannose-Specific Oyster Lectin CGL1.

A novel mannose-specific lectin, named CGL1 (15.5 kDa), was isolated from the oyster Crassostrea gigas. Characterization of CGL1 revealed that it has strict specificity for the mannose monomer and for high mannose-type N-glycans (HMTGs). The primary and crystal structure of CGL1 did not show any homology with known lectins. These characteristics of CGL1 may be helpful as a research tool and for clinical applications. We show a purification protocol of CGL1 from the Pacific oyster.

Purification of AJLec: A Novel Galactose-Specific Lectin from the Sea Anemone Anthopleura japonica.

AJLec (18.5 kDa), a novel galactose-specific lectin, was isolated from the sea anemone Anthopleura japonica. AJLec demonstrates specificity for galactose monomers and ß-linked terminal galactose residues in complex carbohydrates but not for N-acetylgalactosamine (GalNAc) which is commonly recognized by the galactose-binding lectins. Here, we have described the characteristics and process of extraction and purification of AJLec from Anthopleura japonica, which is useful for the analysis of complex carbohydrates.

Novel Ca2+ -independent carbohydrate recognition of the C-type lectins, SPL-1 and SPL-2, from the bivalve Saxidomus purpuratus.

Novel Ca2+ -independent C-type lectins, SPL-1 and SPL-2, were purified from the bivalve Saxidomus purpuratus. They are composed of dimers with either identical (SPL-2 composed of two B-chains) or distinct (SPL-1 composed of A- and B-chains) polypeptide chains, and show affinity for N-acetylglucosamine (GlcNAc)- and N-acetylgalactosamine (GalNAc)-containing carbohydrates, but not for glucose or galactose. A database search for sequence similarity suggested that they belong to the C-type lectin family. X-ray crystallographic analysis revealed definite structural similarities between their subunits and the carbohydrate-recognition domain (CRD) of the C-type lectin family. Nevertheless, these lectins (especially SPL-2) showed Ca2+ -independent binding affinity for GlcNAc and GalNAc. The crystal structure of SPL-2/GalNAc complex revealed that bound GalNAc was mainly recognized via its acetamido group through stacking interactions with Tyr and His residues and hydrogen bonds with Asp and Asn residues, while widely known carbohydrate-recognition motifs among the C-type CRD (the QPD [Gln-Pro-Asp] and EPN [Glu-Pro-Asn] sequences) are not involved in the binding of the carbohydrate. Carbohydrate-binding specificities of individual A- and B-chains were examined by glycan array analysis using recombinant lectins produced from Escherichia coli cells, where both subunits preferably bound oligosaccharides having terminal GlcNAc or GalNAc with α-glycosidic linkages with slightly different specificities.

Identification, Characterization, and X-ray Crystallographic Analysis of a Novel Type of Lectin AJLec from the Sea Anemone Anthopleura japonica.

A novel galactose-specific lectin, AJLec (18.5 kDa), was isolated from the sea anemone, Anthopleura japonica. AJLec was characterized using the hemagglutination assay, isothermal titration calorimetry (ITC), and glycoconjugate microarray analysis and we found that AJLec has a specificity for galactose monomers and ß-linked terminal galactose residues in complex carbohydrates, but not for N-acetylgalactosamine (GalNAc), which is commonly recognized by galactose-binding lectins. The primary structure of AJLec did not show homology with known lectins, and a crystal structural analysis also revealed a unique homodimeric structure. The crystal structure of AJLec complexed with lactose was solved by measuring the sulfur single-wavelength anomalous diffraction (S-SAD) phasing with an in-house Cu Kα source method. This analysis revealed that the galactose residue in lactose was recognized via its O2, O3, and O4 hydroxyl groups and ring oxygen by calcium coordination and two hydrogen bonds with residues in the carbohydrate-binding site, which demonstrated strict specificity for the ß-linked terminal galactose in this lectin.

Carbohydrate recognition by the rhamnose-binding lectin SUL-I with a novel three-domain structure isolated from the venom of globiferous pedicellariae of the flower sea urchin Toxopneustes pileolus.

The globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus contains several biologically active proteins. We have cloned the cDNA of one of the toxin components, SUL-I, which is a rhamnose-binding lectin (RBL) that acts as a mitogen through binding to carbohydrate chains on target cells. Recombinant SUL-I (rSUL-I) was produced in Escherichia coli cells, and its carbohydrate-binding specificity was examined with the glycoconjugate microarray analysis, which suggested that potential target carbohydrate structures are galactose-terminated N-glycans. rSUL-I exhibited mitogenic activity for murine splenocyte cells and toxicity against Vero cells. The three-dimensional structure of the rSUL-I/l-rhamnose complex was determined by X-ray crystallographic analysis at a 1.8 Å resolution. The overall structure of rSUL-I is composed of three distinctive domains with a folding structure similar to those of CSL3, a RBL from chum salmon (Oncorhynchus keta) eggs. The bound l-rhamnose molecules are mainly recognized by rSUL-I through hydrogen bonds between its 2-, 3-, and 4-hydroxy groups and Asp, Asn, and Glu residues in the binding sites, while Tyr and Ser residues participate in the recognition mechanism. It was also inferred that SUL-I may form a dimer in solution based on the molecular size estimated via dynamic light scattering as well as possible contact regions in its crystal structure.

A Single Amino Acid Mutation Converts (R)-5-Diphosphomevalonate Decarboxylase into a Kinase.

The biosynthesis of isopentenyl diphosphate, a fundamental precursor for isoprenoids, via the mevalonate pathway is completed by diphosphomevalonate decarboxylase. This enzyme catalyzes the formation of isopentenyl diphosphate through the ATP-dependent phosphorylation of the 3-hydroxyl group of (R)-5-diphosphomevalonate followed by decarboxylation coupled with the elimination of the 3-phosphate group. In this reaction, a conserved aspartate residue has been proposed to be involved in the phosphorylation step as the general base catalyst that abstracts a proton from the 3-hydroxyl group. In this study, the catalytic mechanism of this rare type of decarboxylase is re-investigated by structural and mutagenic studies on the enzyme from a thermoacidophilic archaeon Sulfolobus solfataricus The crystal structures of the archaeal enzyme in complex with (R)-5-diphosphomevalonate and adenosine 5'-O-(3-thio)triphosphate or with (R)-5-diphosphomevalonate and ADP are newly solved, and theoretical analysis based on the structure suggests the inability of proton abstraction by the conserved aspartate residue, Asp-281. Site-directed mutagenesis on Asp-281 creates mutants that only show diphosphomevalonate 3-kinase activity, demonstrating that the residue is required in the process of phosphate elimination/decarboxylation, rather than in the preceding phosphorylation step. These results enable discussion of the catalytic roles of the aspartate residue and provide clear proof of the involvement of a long predicted intermediate, (R)-3-phospho-5-diphosphomevalonate, in the reaction of the enzyme.

Identification, Characterization, and X-ray Crystallographic Analysis of a Novel Type of Mannose-Specific Lectin CGL1 from the Pacific Oyster Crassostrea gigas.

A novel mannose-specific lectin, named CGL1 (15.5 kDa), was isolated from the oyster Crassostrea gigas. Characterization of CGL1 involved isothermal titration calorimetry (ITC), glycoconjugate microarray, and frontal affinity chromatography (FAC). This analysis revealed that CGL1 has strict specificity for the mannose monomer and for high mannose-type N-glycans (HMTGs). Primary structure of CGL1 did not show any homology with known lectins but did show homology with proteins of the natterin family. Crystal structure of the CGL1 revealed a unique homodimer in which each protomer was composed of 2 domains related by a pseudo two-fold axis. Complex structures of CGL1 with mannose molecules showed that residues have 8 hydrogen bond interactions with O1, O2, O3, O4, and O5 hydroxyl groups of mannose. The complex interactions that are not observed with other mannose-binding lectins revealed the structural basis for the strict specificity for mannose. These characteristics of CGL1 may be helpful as a research tool and for clinical applications.

Effects of amino acid mutations in the pore-forming domain of the hemolytic lectin CEL-III.

The hemolytic lectin CEL-III forms transmembrane pores in the membranes of target cells. A study on the effect of site-directed mutation at Lys405 in domain 3 of CEL-III indicated that replacements of this residue by relatively smaller residues lead to a marked increase in hemolytic activity, suggesting that moderately destabilizing domain 3 facilitates formation of transmembrane pores through conformational changes.

In Vivo Formation of the Protein Disulfide Bond That Enhances the Thermostability of Diphosphomevalonate Decarboxylase, an Intracellular Enzyme from the Hyperthermophilic Archaeon Sulfolobus solfataricus.

UNLABELLED: In the present study, the crystal structure of recombinant diphosphomevalonate decarboxylase from the hyperthermophilic archaeon Sulfolobus solfataricus was solved as the first example of an archaeal and thermophile-derived diphosphomevalonate decarboxylase. The enzyme forms a homodimer, as expected for most eukaryotic and bacterial orthologs. Interestingly, the subunits of the homodimer are connected via an intersubunit disulfide bond, which presumably formed during the purification process of the recombinant enzyme expressed in Escherichia coli. When mutagenesis replaced the disulfide-forming cysteine residue with serine, however, the thermostability of the enzyme was significantly lowered. In the presence of ß-mercaptoethanol at a concentration where the disulfide bond was completely reduced, the wild-type enzyme was less stable to heat. Moreover, Western blot analysis combined with nonreducing SDS-PAGE of the whole cells of S. solfataricus proved that the disulfide bond was predominantly formed in the cells. These results suggest that the disulfide bond is required for the cytosolic enzyme to acquire further thermostability and to exert activity at the growth temperature of S. solfataricus. IMPORTANCE: This study is the first report to describe the crystal structures of archaeal diphosphomevalonate decarboxylase, an enzyme involved in the classical mevalonate pathway. A stability-conferring intersubunit disulfide bond is a remarkable feature that is not found in eukaryotic and bacterial orthologs. The evidence that the disulfide bond also is formed in S. solfataricus cells suggests its physiological importance.

Mannose-recognition mutant of the galactose/N-acetylgalactosamine-specific C-type lectin CEL-I engineered by site-directed mutagenesis.

BACKGROUND: CEL-I is a galactose/N-acetylgalactosamine-specific C-type lectin isolated from the sea cucumber Cucumaria echinata. Its carbohydrate-binding site contains a QPD (Gln-Pro-Asp) motif, which is generally recognized as the galactose specificity-determining motif in the C-type lectins. In our previous study, replacement of the QPD motif by an EPN (Glu-Pro-Asn) motif led to a weak binding affinity for mannose. Therefore, we examined the effects of an additional mutation in the carbohydrate-binding site on the specificity of the lectin. METHODS: Trp105 of EPN-CEL-I was replaced by a histidine residue using site-directed mutagenesis, and the binding affinity of the resulting mutant, EPNH-CEL-I, was examined by sugar-polyamidoamine dendrimer assay, isothermal titration calorimetry, and glycoconjugate microarray analysis. Tertiary structure of the EPNH-CEL-I/mannose complex was determined by X-ray crystallographic analysis. RESULTS: Sugar-polyamidoamine dendrimer assay and glycoconjugate microarray analysis revealed a drastic change in the specificity of EPNH-CEL-I from galactose/N-acetylgalactosamine to mannose. The association constant of EPNH-CEL-I for mannose was determined to be 3.17×10(3) M(-1) at 25°C. Mannose specificity of EPNH-CEL-I was achieved by stabilization of the binding of mannose in a correct orientation, in which the EPN motif can form proper hydrogen bonds with 3- and 4-hydroxy groups of the bound mannose. CONCLUSIONS: Specificity of CEL-I can be engineered by mutating a limited number of amino acid residues in addition to the QPD/EPN motifs. GENERAL SIGNIFICANCE: Versatility of the C-type carbohydrate-recognition domain structure in the recognition of various carbohydrate chains could become a promising platform to develop novel molecular recognition proteins.

cDNA cloning and characterization of a rhamnose-binding lectin SUL-I from the toxopneustid sea urchin Toxopneustes pileolus venom.

The globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus contain several biologically active proteins. Among these, a galactose-binding lectin SUL-I isolated from the venom in the large globiferous pedicellariae shows several activities such as mitogenic, chemotactic, and cytotoxic activities through binding to the carbohydrate chains on the cells. We cloned cDNA encoding SUL-I by reverse transcription-PCR using the degenerate primers designed on the basis of the N-terminal amino acid sequence of the protein and expressed the recombinant SUL-I (rSUL-I) in Escherichia coli cells. The SUL-I gene contains an open reading frame of 927 nucleotides corresponding to 308 amino acid residues, including 24 residues of a putative signal sequence. The mature protein with 284 residues is composed of three homologous regions, each showing similarity with the carbohydrate-recognition domains of the rhamnose-binding lectins, which have been mostly found in fish eggs. While rSUL-I exhibited binding activity for several galactose-related sugars, the highest affinity was found for l-rhamnose among carbohydrates tested, confirming that SUL-I is a rhamnose-binding lectin. rSUL-I also showed hemagglutinating activity toward rabbit erythrocytes, indicating the existence of more than one carbohydrate-binding site to cross-link the carbohydrate chains on the cell surface, which may be closely related to its biological activities.

Structural insights into the low pH adaptation of a unique carboxylesterase from Ferroplasma: altering the pH optima of two carboxylesterases.

To investigate the mechanism for low pH adaptation by a carboxylesterase, structural and biochemical analyses of EstFa_R (a recombinant, slightly acidophilic carboxylesterase from Ferroplasma acidiphilum) and SshEstI (an alkaliphilic carboxylesterase from Sulfolobus shibatae DSM5389) were performed. Although a previous proteomics study by another group showed that the enzyme purified from F. acidiphilum contained an iron atom, EstFa_R did not bind to iron as analyzed by inductively coupled plasma MS and isothermal titration calorimetry. The crystal structures of EstFa_R and SshEstI were determined at 1.6- and 1.5-Å resolutions, respectively. EstFa_R had a catalytic triad with an extended hydrogen bond network that was not observed in SshEstI. Quadruple mutants of both proteins were created to remove or introduce the extended hydrogen bond network. The mutation on EstFa_R enhanced its catalytic efficiency and gave it an alkaline pH optimum, whereas the mutation on SshEstI resulted in opposite effects (i.e. a decrease in the catalytic efficiency and a downward shift in the optimum pH). Our experimental results suggest that the low pH optimum of EstFa_R activity was a result of the unique extended hydrogen bond network in the catalytic triad and the highly negatively charged surface around the active site. The change in the pH optimum of EstFa_R happened simultaneously with a change in the catalytic efficiency, suggesting that the local flexibility of the active site in EstFa_R could be modified by quadruple mutation. These observations may provide a novel strategy to elucidate the low pH adaptation of serine hydrolases.