Kinetic Profile and Molecular Dynamic Studies Show that Y229W Substitution in an NDM-1/L209F Variant Restores the Hydrolytic Activity of the Enzyme toward Penicillins, Cephalosporins, and Carbapenems

ABSTRACT The New Delhi metallo-β-lactamase-1 (NDM-1) enzyme is the most common metallo-β-lactamase identified in many Gram-negative bacteria causing severe nosocomial infections. The aim of this study was to focus the attention on non-active-site residues L209 and Y229 of NDM-1 and to investigate their role in the catalytic mechanism. Specifically, the effect of the Y229W substitution in the L209F variant was evaluated by antimicrobial susceptibility testing, kinetic, and molecular dynamic (MD) studies. The Y229W single mutant and L209F-Y229W double mutant were generated by site-directed mutagenesis. The Km, kcat, and kcat/Km kinetic constants, calculated for the two mutants, were compared with those of (wild-type) NDM-1 and the L209F variant. Compared to the L209F single mutant, the L209F-Y229W double mutant showed a remarkable increase in kcat values of 100-, 240-, 250-, and 420-fold for imipenem, meropenem, benzylpenicillin, and cefepime, respectively. In the L209F-Y229W enzyme, we observed a remarkable increase in kcat/Km of 370-, 140-, and 80-fold for cefepime, meropenem, and cefazolin, respectively. The same behavior was noted using the antimicrobial susceptibility test. MD simulations were carried out on both L209F and L209F-Y229W enzymes complexed with benzylpenicillin, focusing attention on the overall mechanical features and on the differences between the two systems. With respect to the L209F variant, the L209F-Y229W double mutant showed mechanical stabilization of loop 10 and the N-terminal region. In addition, Y229W substitution destabilized both the C-terminal region and the region from residues 149 to 154. The epistatic effect of the Y229W mutation jointly with the stabilization of loop 10 led to a better catalytic efficiency of β-lactams. NDM numbering is used in order to facilitate the comparison with other NDM-1 studies.

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Catalytic and structural contributions for glutathione-binding residues in a Delta class glutathione S-transferase

Biochemical Journal ◽

10.1042/bj20040697 ◽

2004 ◽

Vol 382 (2) ◽

pp. 751-757 ◽

Cited By ~ 23

Author(s):

Pakorn WINAYANUWATTIKUN ◽

Albert J. KETTERMAN

Keyword(s):

Binding Site ◽

Active Site ◽

Structural Integrity ◽

Catalytic Mechanism ◽

Tertiary Structure ◽

Site Directed Mutagenesis ◽

Active Site Residues ◽

Anopheles Dirus ◽

Steady State Kinetics ◽

Cellular Detoxification

Glutathione S-transferases (GSTs) are dimeric proteins that play a major role in cellular detoxification. The GSTs in mosquito Anopheles dirus species B, an important malaria vector in South East Asia, are of interest because they can play an important role in insecticide resistance. In the present study, we characterized the Anopheles dirus (Ad)GST D3-3 which is an alternatively spliced product of the adgst1AS1 gene. The data from the crystal structure of GST D3-3 shows that Ile-52, Glu-64, Ser-65, Arg-66 and Met-101 interact directly with glutathione. To study the active-site function of these residues, alanine substitution site-directed mutagenesis was performed resulting in five mutants: I52A (Ile-52→Ala), E64A, S65A, R66A and M101A. Interestingly, the E64A mutant was expressed in Escherichia coli in inclusion bodies, suggesting that this residue is involved with the tertiary structure or folding property of this enzyme. However, the I52A, S65A, R66A and M101A mutants were purified by glutathione affinity chromatography and the enzyme activity characterized. On the basis of steady-state kinetics, difference spectroscopy, unfolding and refolding studies, it was concluded that these residues: (1) contribute to the affinity of the GSH-binding site (‘G-site’) for GSH, (2) influence GSH thiol ionization, (3) participate in kcat regulation by affecting the rate-limiting step of the reaction, and in the case of Ile-52 and Arg-66, influenced structural integrity and/or folding of the enzyme. The structural perturbations from these mutants are probably transmitted to the hydrophobic-substrate-binding site (‘H-site’) through changes in active site topology or through effects on GSH orientation. Therefore these active site residues appear to contribute to various steps in the catalytic mechanism, as well as having an influence on the packing of the protein.

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Structure based protein engineering to confer selectivity of guanine deaminase

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s205327331409562x ◽

2014 ◽

Vol 70 (a1) ◽

pp. C437-C437

Author(s):

Aruna Bitra ◽

Ruchi Anand

Keyword(s):

Crystal Structures ◽

Active Site ◽

Catalytic Mechanism ◽

Catalytic Efficiency ◽

Structural Basis ◽

Active Site Residues ◽

X Ray ◽

Secondary Activity ◽

Guanine Deaminase ◽

Differential Selectivity

Guanine deaminases (GDs) are important enzymes involved in both purine metabolism and nucleotide anabolism pathways. Here we present the molecular and catalytic mechanism of NE0047 and use the information obtained to engineer specific enzyme activities. NE0047 from Nitrosomonas europaea was found to be a high fidelity guanine deaminase (catalytic efficiency of 1.2 × 105 M–1 s–1). However; it exhibited secondary activity towards the structurally non-analogous triazine based compound ammeline. The X-ray structure of NE0047 in the presence of the substrate analogue 8-azaguanine help establish that the enzyme exists as a biological dimer and both the proper closure of the C-terminal loop and cross talk via the dimeric interface is crucial for conferring catalytic activity. It was further ascertained that the highly conserved active site residues Glu79 and Glu143 facilitate the deamination reaction by serving as proton shuttles. Moreover, to understand the structural basis of dual substrate specificity, X-ray structures of NE0047 in complex with a series of nucleobase analogs, nucleosides and substrate ammeline were determined. The crystal structures demonstrated that any substitutions in the parent substrates results in the rearrangement of the ligand in a catalytically unfavorable orientation and also impede the closure of catalytically important loop, thereby abrogating activity. However, ammeline was able to adopt a catalytically favorable orientation which, also allowed for proper loop closure. Based on the above knowledge of the crystal structures and the catalytic mechanism, the active site was subsequently engineered to fine-tune NE0047 activity. The mutated versions of the enzyme were designed so that they can function either exclusively as a GD or serve as specific ammeline deaminases. For example, mutations in the active site E143D and N66A confer the enzyme to be an unambiguous GD with no secondary activity towards ammeline. On the other hand, the N66Q mutant of NE0047 only deaminates ammeline. Additionally, a series of crystal structures of the mutant versions were solved that shed light on the structural basis of this differential selectivity.

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Characterization of Self-Processing Activities and Substrate Specificities of Porcine Torovirus 3C-Like Protease

Journal of Virology ◽

10.1128/jvi.01282-20 ◽

2020 ◽

Vol 94 (20) ◽

Author(s):

Shangen Xu ◽

Junwei Zhou ◽

Yingjin Chen ◽

Xue Tong ◽

Zixin Wang ◽

...

Keyword(s):

Active Site ◽

Catalytic Mechanism ◽

Site Directed Mutagenesis ◽

Active Site Residues ◽

Hydrophobic Force ◽

Substrate Specificities ◽

Consensus Sequences ◽

Cleavage Activity ◽

Porcine Torovirus

ABSTRACT The 3C-like protease (3CLpro) of nidovirus plays an important role in viral replication and manipulation of host antiviral innate immunity, which makes it an ideal antiviral target. Here, we characterized that porcine torovirus (PToV; family Tobaniviridae, order Nidovirales) 3CLpro autocatalytically releases itself from the viral precursor protein by self-cleavage. Site-directed mutagenesis suggested that PToV 3CLpro, as a serine protease, employed His53 and Ser160 as the active-site residues. Interestingly, unlike most nidovirus 3CLpro, the P1 residue plays a less essential role in N-terminal self-cleavage of PToV 3CLpro. Substituting either P1 or P4 residue of substrate alone has little discernible effect on N-terminal cleavage. Notably, replacement of the two residues together completely blocks N-terminal cleavage, suggesting that N-terminal self-cleavage of PToV 3CLpro is synergistically affected by both P1 and P4 residues. Using a cyclized luciferase-based biosensor, we systematically scanned the polyproteins for cleavage sites and identified (FXXQ↓A/S) as the main consensus sequences. Subsequent homology modeling and biochemical experiments suggested that the protease formed putative pockets S1 and S4 between the substrate. Indeed, mutants of both predicted S1 (D159A, H174A) and S4 (P62G/L185G) pockets completely lost the ability of cleavage activity of PToV 3CLpro. In conclusion, the characterization of self-processing activities and substrate specificities of PToV 3CLpro will offer helpful information for the mechanism of nidovirus 3C-like proteinase’s substrate specificities and the rational development of the antinidovirus drugs. IMPORTANCE Currently, the active-site residues and substrate specificities of 3C-like protease (3CLpro) differ among nidoviruses, and the detailed catalytic mechanism remains largely unknown. Here, porcine torovirus (PToV) 3CLpro cleaves 12 sites in the polyproteins, including its N- and C-terminal self-processing sites. Unlike coronaviruses and arteriviruses, PToV 3CLpro employed His53 and Ser160 as the active-site residues that recognize a glutamine (Gln) at the P1 position. Surprisingly, mutations of P1-Gln impaired the C-terminal self-processing but did not affect N-terminal self-processing. The “noncanonical” substrate specificity for its N-terminal self-processing was attributed to the phenylalanine (Phe) residue at the P4 position in the N-terminal site. Furthermore, a double glycine (neutral) substitution at the putative P4-Phe-binding residues (P62G/L185G) abolished the cleavage activity of PToV 3CLpro suggested the potential hydrophobic force between the PToV 3CLpro and P4-Phe side chains.

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Site-directed mutagenesis of proposed active-site residues of penicillin-binding protein 5 from Escherichia coli

Biochemical Journal ◽

10.1042/bj3030357 ◽

1994 ◽

Vol 303 (2) ◽

pp. 357-362 ◽

Cited By ~ 29

Author(s):

M P G van der Linden ◽

L de Haan ◽

O Dideberg ◽

W Keck

Keyword(s):

Active Site ◽

Catalytic Mechanism ◽

Binding Capacity ◽

Binding Protein ◽

Complete Loss ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Penicillin Binding Protein ◽

Wild Type ◽

Active Site Residues

Alignment of the amino acid sequence of penicillin-binding protein 5 (PBP5) with the sequences of other members of the family of active-site-serine penicillin-interacting enzymes predicted the residues playing a role in the catalytic mechanism of PBP5. Apart from the active-site (Ser44), Lys47, Ser110-Gly-Asn, Asp175 and Lys213-Thr-Gly were identified as the residues making up the conserved boxes of this protein family. To determine the role of these residues, they were replaced using site-directed mutagenesis. The mutant proteins were assayed for their penicillin-binding capacity and DD-carboxypeptidase activity. The Ser44Cys and the Ser44Gly mutants showed a complete loss of both penicillin-binding capacity and DD-carboxypeptidase activity. The Lys47Arg mutant also lost its DD-carboxypeptidase activity but was able to bind and hydrolyse penicillin, albeit at a considerably reduced rate. Mutants in the Ser110-Gly-Asn fingerprint were affected in both acylation and deacylation upon reaction with penicillin and lost their DD-carboxypeptidase activity with the exception of Asn112Ser and Asn112Thr. The Asp175Asn mutant showed wild-type penicillin-binding but a complete loss of DD-carboxypeptidase activity. Mutants of Lys213 lost both penicillin-binding and DD-carboxypeptidase activity except for Lys213His, which still bound penicillin with a k+2/K' of 0.2% of the wild-type value. Mutation of His216 and Thr217 also had a strong effect on DD-carboxypeptidase activity. Thr217Ser and Thr217Ala showed augmented hydrolysis rates for the penicillin acyl-enzyme. This study reveals the residues in the conserved fingerprints to be very important for both DD-carboxypeptidase activity and penicillin-binding, and confirms them to play crucial roles in catalysis.

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Probing the catalytic mechanism of bovine CD38/NAD+glycohydrolase by site directed mutagenesis of key active site residues

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics ◽

10.1016/j.bbapap.2014.03.014 ◽

2014 ◽

Vol 1844 (7) ◽

pp. 1317-1331 ◽

Cited By ~ 6

Author(s):

Isabelle Kuhn ◽

Esther Kellenberger ◽

Céline Cakir-Kiefer ◽

Hélène Muller-Steffner ◽

Francis Schuber

Keyword(s):

Active Site ◽

Catalytic Mechanism ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Active Site Residues ◽

Nad Glycohydrolase

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Structural, Mechanistic, and Functional Insights into an Arthrobacter nicotinovorans Molybdenum Hydroxylase Involved in Nicotine Degradation

Molecules ◽

10.3390/molecules26144387 ◽

2021 ◽

Vol 26 (14) ◽

pp. 4387

Author(s):

Lei Wang ◽

Xia Mu ◽

Wenjin Li ◽

Qin Xu ◽

Ping Xu ◽

...

Keyword(s):

Structural Model ◽

Catalytic Mechanism ◽

Catalytic Efficiency ◽

Degradation Pathway ◽

Site Directed Mutagenesis ◽

Functional Study ◽

Iron Sulfur Clusters ◽

Nicotine Degradation ◽

Arthrobacter Nicotinovorans ◽

Important Enzyme

Arthrobacter nicotinovorans decomposes nicotine through the pyridine pathway. 6-hydroxypseudooxynicotine 2-oxidoreductase (also named ketone dehydrogenase, Kdh) is an important enzyme in nicotine degradation pathway of A. nicotinovorans, and is responsible for the second hydroxylation of nicotine. Kdh belongs to the molybdenum hydroxylase family, and catalyzes the oxidation of 6-hydroxy-pseudooxynicotine (6-HPON) to 2,6-dihydroxy-pseudooxynicotine (2,6-DHPON). We determined the crystal structure of the Kdh holoenzyme from A. nicotinovorans, with its three subunits KdhL, KdhM, and KdhS, and their associated cofactors molybdopterin cytosine dinucleotide (MCD), two iron-sulfur clusters (Fe2S2), and flavin adenine dinucleotide (FAD), respectively. In addition, we obtained a structural model of the substrate 6-HPON-bound Kdh through molecular docking, and performed molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations to unveil the catalytic mechanism of Kdh. The residues Glu345, Try551, and Glu748 of KdhL were found to participate in substrate binding, and Phe269 and Arg383 of KdhL were found to contribute to stabilize the MCD conformation. Furthermore, site-directed mutagenesis and enzymatic activity assays were performed to support our structural and computational results, which also revealed a trend of increasing catalytic efficiency with the increase in the buffer pH. Lastly, our electrochemical results demonstrated electron transfer among the various cofactors of Kdh. Therefore, our work provides a comprehensive structural, mechanistic, and functional study on the molybdenum hydroxylase Kdh in the nicotine degradation pathway of A. nicotinovorans.

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Site-directed mutagenesis reveals a novel catalytic mechanism of Mycobacterium tuberculosis alkylhydroperoxidase C

Biochemical Journal ◽

10.1042/bj20020545 ◽

2002 ◽

Vol 367 (1) ◽

pp. 255-261 ◽

Cited By ~ 32

Author(s):

Radha CHAUHAN ◽

Shekhar C. MANDE

Keyword(s):

Mycobacterium Tuberculosis ◽

Reaction Mechanism ◽

Kinetic Parameters ◽

Active Site ◽

Double Mutant ◽

Catalytic Mechanism ◽

Site Directed Mutagenesis ◽

Cysteine Residues ◽

Directed Mutagenesis ◽

Resistant Strains

Mycobacterium tuberculosis alkylhydroperoxidase C (AhpC) belongs to the peroxiredoxin family, but unusually contains three cysteine residues in its active site. It is overexpressed in isoniazid-resistant strains of M. tuberculosis. We demonstrate that AhpC is capable of acting as a general antioxidant by protecting a range of substrates including supercoiled DNA. Active-site Cys to Ala mutants show that all three cysteine residues are important for activity. Cys-61 plays a central role in activity and Cys-174 also appears to be crucial. Interestingly, the C174A mutant is inactive, but double mutant C174/176A shows significant revertant activity. Kinetic parameters indicate that the C176A mutant is active, although much less efficient. We suggest that M. tuberculosis AhpC therefore belongs to a novel peroxiredoxin family and might follow a unique disulphide-relay reaction mechanism.

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Amino Acids Asp695 and Tyr696 of Factor Va Are Essential for Optimal Rate of Prothrombin Activation by Prothrombinase.

Blood ◽

10.1182/blood.v114.22.3163.3163 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3163-3163

Author(s):

Jamila Hirbawi ◽

Michael Kalafatis

Keyword(s):

Amino Acids ◽

Heavy Chain ◽

Conflicts Of Interest ◽

Catalytic Efficiency ◽

Terminal Region ◽

Site Directed Mutagenesis ◽

Prothrombinase Complex ◽

Factor Va ◽

Sds Page ◽

Prothrombin Activation

Abstract Abstract 3163 Poster Board III-101 Blood coagulation is initiated after vascular injury, promoting formation of the fibrin clot. Without the proper regulation of this process, serious life threatening conditions, such as DVT (deep vein thrombosis), can occur. The proteolytic conversion of prothrombin to thrombin is catalyzed by the prothrombinase complex composed of the enzyme, factor Xa (fXa), the cofactor, factor Va (fVa), assembled on a membrane surface in the presence of Ca2+. The incorporation of fVa into the prothrombinase complex results in a 300,000-fold increase in the catalytic efficiency of fXa for thrombin generation. Prothrombinase activates prothrombin through initial cleavage at Arg320 followed by cleavage at Arg271 to yield human alpha-thrombin. This pathway is responsible for the generation of a transient intermediate, meizothrombin, that is enzymatically active with increased chromogenic substrate activity, but yields poor clotting activity. Factor Va is composed of heavy and light chains that play a crucial role during thrombin formation. Portions of the fVa heavy chain have been found to interact with proexosite 1 (pro1) of prothrombin and influence prothrombinase activity. It has been recently demonstrated that deletion of the COOH-terminal region of the factor Va heavy chain causes accumulation of meizothrombin due to delayed cleavage of prothrombin at Arg271. Site-directed mutagenesis was performed to generate recombinant mutant molecules in order to identify the specific amino acids of this COOH-terminal region that regulate cleavage. Mutants with the 695DYDY698→DFDY(fVaDFDY), KFDY(fVaKFDY),DEDE(fVaDEDE),DFDF(fVaDFDF) substitutions were constructed. These recombinant molecules along with wild type factor V (fVWT) were transiently expressed in COS7 cells purified to homogeneity and assessed for their capability to promote prothrombin activation. Prothrombin activation was evaluated by SDS-PAGE and the kinetic parameters of the reactions were determined. SDS-PAGE analyses of prothrombin activation time courses revealed that the overall cleavage of prothrombin by prothrombinase assembled with fVaKFDY and fVaDFDY was delayed, while prothrombinase assembled with fVaDEDE and fVaDFDF had no significant effects when compared to fVaWT. Two- stage clotting assays (PT times) revealed that fVaKFDY and fVaDFDY both had reduced clotting activity when compared to fVaWT, while fVaDEDE and fVaDFDF gave similar clotting results as fVaWT. Determination of kcat values for prothrombinase assembled with the various recombinant molecules revealed that prothrombinase assembled fVaKFDY and fVaDFDY had a 20% increase catalytic efficiency as compared with prothrombinase assembled with fVaWT, while fVaDEDE and fVaDFDF gave values that were comparable to prothrombinase assembled with fVaWT. Comparison of the rate of cleavage of two recombinant prothrombin mutant molecules, rMZ that can't be cleaved at Arg271 and rPII that can't be cleaved at Arg320, by prothrombinase assembled with the mutant fVa molecules resulted in normal cleavage of rMZ by all the mutants. Cleavage of rPII, however, was impaired when fVaKFDY and fVaDFDY were incorporated into prothrombinase. The data presented suggests that the 695DY696 portion of the acidic cluster found in the COOH-terminus of the fVa heavy chain plays a significant role in enzyme-substrate interaction during thrombus formation. Disclosures No relevant conflicts of interest to declare.

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A Novel Glycoside Hydrolase Family 113 Endo-β-1,4-Mannanase from Alicyclobacillus sp. Strain A4 and Insight into the Substrate Recognition and Catalytic Mechanism of This Family

Applied and Environmental Microbiology ◽

10.1128/aem.04071-15 ◽

2016 ◽

Vol 82 (9) ◽

pp. 2718-2727 ◽

Cited By ~ 14

Author(s):

Wei Xia ◽

Haiqiang Lu ◽

Mengjuan Xia ◽

Ying Cui ◽

Yingguo Bai ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Catalytic Mechanism ◽

Substrate Recognition ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Glycoside Hydrolase Family ◽

Content Type ◽

Binding Pockets ◽

Alicyclobacillus Acidocaldarius ◽

Key Residues

ABSTRACTFew members of glycoside hydrolase (GH) family 113 have been characterized, and information on substrate recognition by and the catalytic mechanism of this family is extremely limited. In the present study, a novel endo-β-1,4-mannanase of GH 113, Man113A, was identified in thermoacidophilicAlicyclobacillussp. strain A4 and found to exhibit both hydrolytic and transglycosylation activities. The enzyme had a broad substrate spectrum, showed higher activities on glucomannan than on galactomannan, and released mannobiose and mannotriose as the main hydrolysis products after an extended incubation. Compared to the only functionally characterized and structure-resolved counterpartAlicyclobacillus acidocaldariusManA (AaManA) of GH 113, Man113A showed much higher catalytic efficiency on mannooligosaccharides, in the order mannohexaose ≈ mannopentaose > mannotetraose > mannotriose, and required at least four sugar units for efficient catalysis. Homology modeling, molecular docking analysis, and site-directed mutagenesis revealed the vital roles of eight residues (Trp13, Asn90, Trp96, Arg97, Tyr196, Trp274, Tyr292, and Cys143) related to substrate recognition by and catalytic mechanism of GH 113. Comparison of the binding pockets and key residues of β-mannanases of different families indicated that members of GH 113 and GH 5 have more residues serving as stacking platforms to support −4 to −1 subsites than those of GH 26 and that the residues preceding the acid/base catalyst are quite different. Taken as a whole, this study elucidates substrate recognition by and the catalytic mechanism of GH 113 β-mannanases and distinguishes them from counterparts of other families.

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Catalytic mechanism and evolutionary characteristics of thioredoxin from Halobacterium salinarum NRC-1

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798319015894 ◽

2020 ◽

Vol 76 (1) ◽

pp. 73-84

Author(s):

Shigeki Arai ◽

Chie Shibazaki ◽

Rumi Shimizu ◽

Motoyasu Adachi ◽

Matsujiro Ishibashi ◽

...

Keyword(s):

Active Site ◽

Catalytic Mechanism ◽

Chain Structure ◽

Crystallographic Analysis ◽

Terminal Region ◽

High Salt ◽

Halobacterium Salinarum ◽

Molecular Surface ◽

Active Site Residues ◽

Potential Applications

Thioredoxin (TRX) is an important antioxidant against oxidative stress. TRX from the extremely halophilic archaeon Halobacterium salinarum NRC-1 (HsTRX-A), which has the highest acidic residue content [(Asp + Glu)/(Arg + Lys + His) = 9.0] among known TRXs, was chosen to elucidate the catalytic mechanism and evolutionary characteristics associated with haloadaptation. X-ray crystallographic analysis revealed that the main-chain structure of HsTRX-A is similar to those of homologous TRXs; for example, the root-mean-square deviations on Cα atoms were <2.3 Å for extant archaeal TRXs and <1.5 Å for resurrected Precambrian TRXs. A unique water network was located near the active-site residues (Cys45 and Cys48) in HsTRX-A, which may enhance the proton transfer required for the reduction of substrates under a high-salt environment. The high density of negative charges on the molecular surface (3.6 × 10−3 e Å−2) should improve the solubility and haloadaptivity. Moreover, circular-dichroism measurements and enzymatic assays using a mutant HsTRX-A with deletion of the long flexible N-terminal region (Ala2–Pro17) revealed that Ala2–Pro17 improves the structural stability and the enzymatic activity of HsTRX-A under high-salt environments (>2 M NaCl). The elongation of the N-terminal region in HsTRX-A accompanies the increased hydrophilicity and acidic residue content but does not affect the structure of the active site. These observations offer insights into molecular evolution for haloadaptation and potential applications in halophilic protein-related biotechnology.

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