Structure-function relationships in human lecithin:cholesterol acyltransferase. Site-directed mutagenesis at serine residues 181 and 216

The functions of serine residues at positions 181 and 216 of human plasma lecithin:cholesterol acyltransferase have been studied by site-directed mutagenesis. The serine residue at either site was replaced by alanine, glycine, or threonine in LCAT secreted from stably transfected CHO cells. All substitutions at position 181 gave rise to an enzyme product that was normally secreted but had no detectable catalytic activity. On the other hand, all substitutions at position 216 gave active products, whose activity was fully inhibitable by the serine esterase inhibitor diisopropyl fluorophosphate (DFP). A secondary (although not direct) role for serine-216 was indicated by a 14-fold increase in catalytic rate when this residue was substituted by alanine. Sequence comparison with other lipases suggests that serine-216 may be at or near the hinge of a helical flap displaced following substrate binding. These data strengthen the structural-functional relationship between LCAT and other lipases.     

Neisseria gonorrheae O-acetylpeptidoglycan esterase, a serine esterase with a Ser-His-Asp catalytic triad

O-Acetylpeptidoglycan esterase from Neisseria gonorrheae FA1090 is similar in sequence to family CE-3 carbohydrate esterases of the CAZy classification system, and it functions to release O-linked acetyl groups from the C-6 position of muramoyl residues in O-acetylated peptidoglycan. Here, we characterize the peptidoglycan of N. gonorrheae FA1090 as being O-acetylated and find that it serves as a substrate for the esterase. The influence of pH on the activity of O-acetylpeptidoglycan esterase was determined, and pKa values of 6.38 and 6.78 for the enzyme-substrate complex (VEt-1) and free enzyme (VEt-1KM-1), respectively, were calculated. The enzyme was inactivated by sulfonyl fluorides but not by EDTA. Multiple-sequence alignment of the O-acetylpeptidoglycan esterase family 1 enzymes with members of the CE-3 enzymes and protein modeling studies identified Ser80, Asp366, and His369 as three invariant amino acid residues that could potentially serve as a catalytic triad. Replacement of each with alanine was accomplished by site-directed mutagenesis, and the resulting mutant proteins were purified to apparent homogeneity. The specific activity of each of the three esterase derivatives was greatly reduced on O-acetylpeptidoglycan. Using the artificial substrate p-nitrophenyl acetate, a kinetic analysis revealed that the turnover number (VEt-1) but not KM was affected by the replacements. These data thus indicate that N. gonorrheae O-acetylpeptidoglycan esterase, and by analogy the CE-3 family of enzymes, function as serine esterases involving a Ser-His-Asp catalytic triad.     

Site-specific mutagenesis of an essential histidine residue in pancreatic cholesterol esterase

The histidine residue essential for the catalytic activity of pancreatic cholesterol esterase (carboxylester lipase) has been identified in this study using sequence comparison and site-specific mutagenesis techniques. In the first approach, comparison of the primary structure of rat pancreatic cholesterol esterase with that of acetylcholinesterase and cholinesterase revealed two conserved histidine residues located at positions 420 and 435. The sequence in the region around histidine 420 is quite different between the three enzymes. However, histidine 435 is located in a 22-amino acid domain that is 47% homologous with other serine esterases. Based on this sequence homology, it was hypothesized that histidine 435 is the histidine residue essential for catalytic activity of cholesterol esterase. The role of His435 in the catalytic activity of pancreatic cholesterol esterase was then studied by the site-specific mutagenesis technique. Substitution of the histidine in position 435 with glutamine, arginine, alanine, serine, or aspartic acid abolished the ability of cholesterol esterase to hydrolyze p-nitrophenyl butyrate and cholesterol [14C]oleate. In contrast, mutagenesis of the histidine residue at position 420 to glutamine had no effect on cholesterol esterase enzyme activity. The results of this study strongly suggested that histidine 435 may be a component of the catalytic triad of pancreatic cholesterol esterase.     

A new carboxylesterase from Brevibacterium linens IFO 12171 responsible for the conversion of 1,4-butanediol diacrylate to 4-hydroxybutyl acrylate: purification, characterization, gene cloning, and gene expression in Escherichia coli.

A carboxylesterase that is responsible for conversion of 1,4-butanediol diacrylate (BDA) to 4-hydroxybutyl acrylate (4HBA) was found in Brevibacterium lines IFO 12171, and purified to homogeneity. The purified enzyme was active toward a variety of diesters of ethylene glycol, 1,4-butanediol, and 1,6-hexanediol. The K(m) and kcat of the enzyme for BDA were 3.04 mM and 203,000 s-1, respectively. The reaction with the purified enzyme gave 98 mM 4HBA from 100 mM BDA for 60 min. The enzyme gene was cloned from the chromosomal DNA of the bacterium. The open reading frame encoding the enzyme was 1176 bp long, corresponding to a protein of 393 amino acid residues (molecular mass = 42,569 Da). The deduced amino acid sequence contained the tetra peptide motif sequence, STTK, and the serine residue was confirmed to be the catalytic center of BDA esterase by site-directed mutagenesis for several amino acid residues. The gene was expressed in Escherichia coli under the control of the lac promoter, and the gene product (a fusion protein with 6 amino acid residues from beta-galactosidase) showed the same catalytic properties as the enzyme from the parent strain.     

Identification of the active site residues in dipeptidyl peptidase IV by affinity labeling and site-directed mutagenesis.

The active site of dipeptidyl peptidase IV (DPPIV) was examined by chemical modification and site-directed mutagenesis. Purified DPPIV was covalently modified with [3H]diisopropyl fluorophosphate (DFP). The radiolabeled DPPIV was digested with lysyl endopeptidase, and the peptides were separated by high-performance liquid chromatography. A single 3H-containing peptide was obtained and analyzed for amino acid sequence and radioactivity distribution. A comparison of the determined sequence with the predicted primary structure of DPPIV [Ogata, S., Misumi, Y., & Ikehara, Y. (1989) J. Biol. Chem. 264, 3596-3601] revealed that [3H]DFP was bound to Ser631 within the sequence Gly629-Trp-Ser-Tyr-Gly633, which corresponds to the consensus sequence Gly-X-Ser-X-Gly proposed for serine proteases. To further identify the essential residues in the active-site sequence, we modified the DPPIV cDNA by site-directed mutagenesis to encode its variants. Expression of the mutagenized cDNAs in COS-1 cells demonstrated that any single substitution of Gly629, Ser631, or Gly633 with other residues resulted in the complete loss of the enzyme activity and DFP binding. Although substitution of Trp630----Glu or Tyr632----Phe caused no effect on the enzyme activity, that of Tyr632----Leu or Gly abolished the activity. These results indicate that the sequence Gly-X-Ser-(Tyr)-Gly is essential for the expression of the DPPIV activity.     

Enantioselectivity of Candida rugosa lipases (Lip1, Lip3, and Lip4) towards 2-bromo phenylacetic acid octyl esters controlled by a single amino acid

Enantiomer discrimination by enzymes is a very accurate mechanism, which often involves few amino acids located at the active site. Lipase isoforms from Candida rugosa are very good enzymatic models to study this phenomenon as they display high sequence homology (>80%) and their enantioselectivity is often pointed out. In the present work, we investigated three lipases from C. rugosa (Lip1, Lip3, and Lip4, respectively) towards the resolution of 2-bromo-arylacetic acid esters, an important class of chemical intermediates in the pharmaceutical industry. All exhibited a high enantioselectivity, with Lip4 preferring the R-enantiomer (E-value = 15), while Lip1 and Lip3 showed an S-enantioselectivity >200. A combination of sequence and structure analysis of the three C. rugosa lipases suggested that position 296 could play a role in S- or R-enantiomer preference of C. rugosa lipases. This led to the construction by site-directed mutagenesis of Lip1 and Lip4 variants in which position 296 was, respectively, exchanged by a Gly, Ala, Leu, or Phe amino acid. Screening of these variants for their enantioselectivity toward 2-bromo phenyl acetic acid octyl esters revealed that steric hindrance of the amino acid residue introduced at position 296 controls both the enantiopreference and the enantioselectivity value of the lipase: bulkier is the amino acid at position 296, larger is the selectivity towards the S-enantiomer. To investigate further these observations at an atomic level, we carried out a preliminary modeling study of the tetrahedral intermediates formed by Lip1 and Lip4 with the (R, S)-2-bromo-phenylacetic acid octyl ester enantiomers that provides some insight regarding the determinants responsible for lipase enantiodiscrimination.     

Simultaneous determination of a selective adenosine 2A agonist, BMS-068645, and its acid metabolite in human plasma by liquid chromatography-tandem mass spectrometry--evaluation of the esterase inhibitor, diisopropyl fluorophosphate, in the stabilization of a labile ester-containing drug

BMS-068645 is a selective adenosine 2A agonist that contains a methyl ester group which undergoes esterase hydrolysis to its acid metabolite. To permit accurate determinations of circulating BMS-068645 and its acid metabolite, blood samples must be rapidly stabilized at the time of collection. A sensitive, rapid and specific liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the simultaneous quantitation of BMS-068645 and its acid metabolite in human plasma has been developed and validated using diisopropyl fluorophosphate (DFP) as the esterase inhibitor to prevent BMS-068645 from converting to its acid metabolite. The D(5)-stable isotope labeled analogs of BMS-068645 and its metabolite were used as the internal standards (IS). Analytes and IS in plasma containing 20 mM DFP were acidified and extracted into methyl tert-butyl ether. The liquid-liquid extraction effectively eliminated the strong matrix effect caused by the esterase inhibitor. The chromatographic separation was achieved on a Waters Atlantis C18 column with a run time of 4 min. Detection was performed on a Sciex API 4000 with positive ion electrospray mode (ESI/MS/MS), monitoring the ion transitions m/z 487>314 and 473>300 for BMS-068645 and its acid metabolite, respectively. The method was validated over the range from 0.020 to 10.0 ng/mL for BMS-068645 and 0.050 to 10.0 ng/mL for its acid metabolite. Inter- and intra-run precision for the quality control samples during validation were less than 8.7% and 4.0%, respectively, for the two analytes. The assay accuracy was within +/-5.4% of the nominal values. The esterase inhibitor effectively stabilized BMS-068645 during blood collection and storage. Blood collection tubes containing DFP were easily prepared and used at the clinical sites and could be stored at -30 degrees C for 3 months. This method demonstrated adequate sensitivity, specificity, accuracy, precision, stability and ruggedness to support the analysis of human plasma samples in pharmacokinetic studies.     

Tryptophan-216 is essential for the transglycosylation activity of endo-beta-N-acetylglucosaminidase A

Endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) has a high level of transglycosylation activity. To determine which amino acids are involved in this activity, we employed deletion analysis, as well as random and site-directed mutagenesis. Using PCR random mutagenesis, 11 mutants with greatly decreased levels of enzyme activity were isolated. Six catalytically essential amino acids were identified by site-directed mutagenesis. Mutants E173G, E175Q, D206G, and D270N had markedly reduced hydrolysis activity, while mutants V109D, E173D, and E173Q lost all enzymatic activity, indicating that Val-109 and Glu-173 are important for the catalytic function. Moreover, we isolated a random mutation that abolished the transglycosylation activity without affecting the hydrolysis activity. The Trp-216 to Arg mutation was identified, by site-directed mutagenesis, as that responsible for the loss of transglycosylation activity. While other mutants of Trp-216 showed reduced activity, mutation to another positively charged residue (Lys) also abolished the transglycosylation activity. Sequence comparison with two other endo-beta-N-acetylglucosaminidases, that possess transglycosylation activity and that have been cloned recently, reveals a high degree of identity in the N-terminal regions of the three enzymes. These results indicate that the tryptophan residue at position 216 of Endo-A has a key role in the transglycosylation.     

Identification of the active site serine in pancreatic cholesterol esterase by chemical modification and site-specific mutagenesis

Chemical modification and site-specific mutagenesis approaches were used in this study to identify the active site serine residue of pancreatic cholesterol esterase. In the first approach, purified porcine pancreatic cholesterol esterase was covalently modified by incubation with [3H]diisopropylfluorophosphate (DFP). The radiolabeled cholesterol esterase was digested with CNBr, and the peptides were separated by high performance liquid chromatography. A single 3H-containing peptide was obtained for sequence determination. The results revealed the binding of DFP to a serine residue within the serine esterase homologous domain of the protein. Furthermore, the DFP-labeled serine was shown to correspond to serine residue 194 of rat cholesterol esterase (Kissel, J. A., Fontaine, R. N., Turck, C. W., Brockman, H. L., and Hui, D. Y. (1989) Biochim. Biophys. Acta 1006, 227-236). The codon for serine 194 in rat cholesterol esterase cDNA was then mutagenized to ACT or GCT to yield mutagenized cholesterol esterase with either threonine or alanine, instead of serine, at position 194. Expression of the mutagenized cDNA in COS-1 cells demonstrated that substitution of serine 194 with threonine or alanine abolished enzyme activity in hydrolyzing the water-soluble substrate, p-nitrophenyl butyrate, and the lipid substrates cholesteryl [14C]oleate and [14C] lysophosphatidylcholine. These studies definitively identified serine 194 in the catalytic site of pancreatic cholesterol esterase.     

Role of cysteine residues in esterase from Bacillus stearothermophilus and increasing its thermostability by the replacement of cysteines

Bacillus stearothermophilus esterase contains two free cysteine residues at positions of 45 and 115, which react with sulfhydryl reagents resulting in a significant decrease in the enzymatic activity. To understand the role of the cysteine residues in catalytic regions of the esterase, the residues were replaced with serine or alanine by site-directed mutagenesis to construct four single-mutated enzymes (C45A, C45S, C115A, C115S) and two double-mutated ones (C45/115A and C45/115S). Wild-type and mutant enzymes were produced in Escherichia coli cells and purified to homogeneity to examine their chemical and kinetic properties. These mutant enzymes had esterase activity, which suggested that none of the cysteines were required for its activity. Moreover, replacement of both two-cysteine residues made the enzyme insensitive to p-chloromercuribenzoic acid and extensively stabilized it at high temperatures of around 70°C. These results demonstrate that replacement of free cysteine residues by site-directed mutagenesis can improve the thermostability of thermophilic enzymes. Correspondence to: T. Yamane     

A novel esterase from Burkholderia gladioli which shows high deacetylation activity on cephalosporins is related to beta-lactamases and DD-peptidases

The gene (estB) encoding for a novel esterase (EstB) from Burkholderia gladioli (formerly Pseudomonas marginata) NCPPB 1891 was cloned in Escherichia coli. Sequence analysis showed an open reading frame encoding a polypeptide of 392 amino acid residues, with a molecular mass of about 42 kDa. Comparison of the amino acid sequence with those of other homologous enzymes indicated homologies to beta-lactamases, penicillin binding proteins and DD-peptidases. The serine residue (Ser(75)) which is located within a present class A beta-lactamase motif ([F,Y]-X-[L,I,V,M,F,Y]-X-S-[T,V]-X-K-X-X-X-X-[A,G,L]-X-X-[L,C]) was identified by site-directed mutagenesis to represent the active nucleophile. A second serine residue (Ser(149)) which is located within a G-x-S-x-G motif which is typically found in esterases and lipases was demonstrated not to play a significant role in enzyme function. The estB gene was overexpressed in E. coli using a tac promoter-based expression system. Investigation of EstB protein with respect to the ability to hydrolyse beta-lactam substrates clearly demonstrated that this protein has no beta-lactamase activity. The recombinant enzyme is active on triglycerides and on nitrophenyl esters with acyl chain lengths up to C6. The preference for short chain length substrates indicated that EstB is a typical carboxylesterase. As a special feature EstB esterase was found to have high deacetylation activity on cephalosporin derivatives.     

Interaction of site specific hirudin variants with alpha-thrombin

The kinetics of complex formation between recombinant hirudin or recombinant hirudin mutants with thrombin were analyzed. In order to elucidate the inhibitor's reactive site peptide bond predetermined amino acid substitutions were introduced at positions of basic amino acid residues by means of site-directed mutagenesis of a hirudin gene. In comparison to recombinant hirudin (Ki = 19 pM) only those mutant inhibitors which were modified at amino acid position Lys47 showed a higher Ki value for their complexes with thrombin. The observed effects are mainly due to increased koff rate constants.     

Isolation and characterization of a thermostable esterase from a metagenomic library

A novel esterase gene was isolated by functional screening of a metagenomic library prepared from an activated sludge sample. The gene (est-XG2) consists of 1,506 bp with GC content of 74.8 %, and encodes a protein of 501 amino acids with a molecular mass of 53 kDa. Sequence alignment revealed that Est-XG2 shows a maximum amino acid identity (47 %) with the carboxylesterase from Thermaerobacter marianensis DSM 12885 (YP_004101478). The catalytic triad of Est-XG2 was predicted to be Ser₁₉₂-Glu₃₁₃-His₄₁₂ with Ser₁₉₂ in a conserved pentapeptide (GXSXG), and further confirmed by site-directed mutagenesis. Phylogenetic analysis suggested Est-XG2 belongs to the bacterial lipase/esterase family VII. The recombinant Est-XG2, expressed and purified from Escherichia coli, preferred to hydrolyze short and medium length p-nitrophenyl esters with the best substrate being p-nitrophenyl acetate (K _m and k _cat of 0.33 mM and 36.21 s^?1, respectively). The purified enzyme also had the ability to cleave sterically hindered esters of tertiary alcohols. Biochemical characterization of Est-XG2 revealed that it is a thermophilic esterase that exhibits optimum activity at pH 8.5 and 70 °C. Est-XG2 had moderate tolerance to organic solvents and surfactants. The unique properties of Est-XG2, high thermostability and stability in the presence of organic solvents, may render it a potential candidate for industrial applications.     

Enhanced bacteriolytic activity of hen egg-white lysozyme due to conversion of Trp62 to other aromatic amino acid residues

Tryptophan at the 62nd position (Trp62) of hen egg-white lysozyme is an amino acid residue whose action is essential for its enzymatic activity. Its indole ring may possibly come into direct contact with sugar residues of the substrate, and thus contribute significantly to substrate binding. For further elucidation of its role in catalytic processes, this amino acid was converted to other aromatic residues, such as Tyr, Phe, and His, by site-directed mutagenesis. All the mutations were found to enhance the bacteriolytic activity but to decrease the hydrolytic activity toward an artificial substrate, glycol chitin. Such a change in substrate preference appears remarkable considering the smaller size of the aromatic residue on the mutant enzyme at the 62nd position.     

Inverting enantioselectivity of Burkholderia gladioli esterase EstB by directed and designed evolution

Esterase EstB from Burkholderia gladioli, showing moderate S-enantioselectivity (E(S)=6.1) in the hydrolytic kinetic resolution of methyl-beta-hydroxyisobutyrate, was subjected to directed evolution in order to reverse its enantioselectivity. After one round of ep-PCR, saturation mutagenesis and high-throughput screening, it was found that different mutations at position 152 (in the vicinity of the active site) increase, decrease and even reverse the natural enantioselectivity of this enzyme. The newly created R-enantioselectivity of the esterase mutein (E(Rapp)=1.5) has been further enhanced by a designed evolution strategy involving random mutations close to the active site. Based on the three-dimensional structure nineteen amino acid residues have been selected as mutation sites for saturation mutagenesis. Mutations at three sites (135, 253 and 351) were found to increase R-enantioselectivity. Successive rounds of saturation mutagenesis at these "hot spots" resulted in an increase in R-enantioselectivity from E(Rapp)=1.5 for the parent mutant to E(Rapp)=28.9 for the best variant which carried four amino acid substitutions. Our results prove designed evolution followed by high-throughput screening to be an efficient strategy for engineering enzyme enantioselectivity.     

Site-directed mutagenesis of Aspergillus niger NRRL 3135 phytase at residue 300 to enhance catalysis at pH 4.0

Increased phytase activity for Aspergillus niger NRRL 3135 phytaseA (phyA) at intermediate pH levels (3.0-5.0) was achieved by site-directed mutagenesis of its gene at amino acid residue 300. A single mutation, K300E, resulted in an increase of the hydrolysis of phytic acid of 56% and 19% at pH 4.0 and 5.0, respectively, at 37 degrees C. This amino acid residue has previously been identified as part of the substrate specificity site for phyA and a comparison of the amino acid sequences of other cloned fungal phytases indicated a correlation between a charged residue at this position and high specific activity for phytic acid hydrolysis. The substitution at this residue by either another basic (R), uncharged (T), or acidic amino acid (D) did not yield a recombinant enzyme with the same favorable properties. Therefore, we conclude that this residue is not only important for the catalytic function of phyA, but also essential for imparting a favorable pH environment for catalysis.     

The requirement for esterase activation in the anti-immunoglobulin-triggered movement of B lymphocytes.

In this paper we bring evidence suggesting that there is activation of an esterase upon reaction of anti-immunoglobulin antibodies (anti-Ig) with murine B lymphocytes. B lymphocytes upon exposure to anti-Ig cap the ligand-receptor complexes and immediately afterward become briefly motile. It is this latter step which is inhibitable by exposure to di-isopropyl phosphofluoridate (DFP). Various experimental manipulations indicated that treatment with anti-Ig activates the cell for motility which, however, is not manifested until the temperature is raised to 37 degrees C. The cell incubated with anti-Ig at cold temperatures becomes susceptible to the effect of DFP, suggesting that the antibody-treated cells are activated up to but not beyond the DFP inhibitable step. Exposure of cells to DFP and removal of it before their treatment with anti-Ig does not affect the anti-Ig-induced response. Four lines of evidence indicate that the reduction of lymphocyte movement of DFP is due to the inhibition of an esterase activated by the combination of antibody and cell: 1) The inhibition by DFP is irreversible; once DFP has reacted it can be washed away and the antibody-treated cell is still inhibited. 2) The inhibition increases with time of contact of lymphocytes and DFP and with the concentration of DFP. 3) A very poorly phosphorylating phosphonate, phenyl ethyl pentylphosphonate is completely inactive under conditions where an excellent phosphorylating phosphonate, p nitrophenyl ethyl pentylphosphonate maximally inactivated the cells' movement. 4) The amino acid esters, tosyl L arginine methyl ester and benzoyl arginine methyl ester specifically prevent the inactivation by DFP. The last finding suggests that tosyl L arginine methyl ester and benzoyl L arginine methyl ester might be substrates for the putative antibody-induced lymphocyte esterase. Lymphocytes incubated with antibody in the cold for more than 30 min lose their ability to move when the temperature is raised, suggesting that there is a time-dependent deactivation of the cell.     

Sequence determinants of 3A-mediated resistance to enviroxime in rhinoviruses and enteroviruses.

Using site-directed mutagenesis of the 3A coding region of rhinovirus 14, we have expanded our analysis of resistance to enviroxime. We have observed that high and low levels of drug resistance involve two domains within 3A and that the amino acid at position 30 is critical in determining resistance.     

Evidence that the amino acid residue P272 of arginine kinase is involved in its activity, structure and stability

In this research, the role of amino acid residue P272 of arginine kinase (AK) was investigated by site-directed mutagenesis. When the structure of AK was impaired by mutation, AK was in a partially unfolded state with more hydrophobic exposure, which was prone to aggregate under environmental stresses. Mutation at this position influences transition from the molten globule intermediate to the native state in folding process. The results provided herein may suggest that some residues near the active site may play a relatively important role in keeping AK activity and structural stability.