Non-heme Peroxidase. Bacterial and fungi non-heme bromo- and chloro-peroxidases family. Haloperoxidases catalyze the halogenation of organic compounds in the presence of halide ions and peroxides such as H2O2. They are named after the most electronegative halide they are able to oxidize and are classified according to their cofactor dependence as heme-type, vanadium-dependent and metal-free haloperoxidases (WARNING Only metal free haloperoxidases are alpha beta hydrolases with a Ser-His-Asp catalytic triad in the active center. This family contains also homologous arylesterases.)
Acyl transfer is a key reaction in biosynthesis, including synthesis of antibiotics and polyesters. Although researchers have long recognized the similar protein fold and catalytic machinery in acyltransferases and hydrolases, the molecular basis for the different reactivity has been a long-standing mystery. By comparison of X-ray structures, we identified a different oxyanion-loop orientation in the active site. In esterases/lipases a carbonyl oxygen points toward the active site, whereas in acyltransferases a NH of the main-chain amide points toward the active site. Amino acid sequence comparisons alone cannot identify such a difference in the main-chain orientation. To identify how this difference might change the reaction mechanism, we solved the X-ray crystal structure of Pseudomonas fluorescens esterase containing a sulfonate transition-state analogue bound to the active-site serine. This structure mimics the transition state for the attack of water on the acyl-enzyme and shows a bridging water molecule between the carbonyl oxygen mentioned above and the sulfonyl oxygen that mimics the attacking water. A possible mechanistic role for this bridging water molecule is to position and activate the attacking water molecule in hydrolases, but to deactivate the attacking water molecule in acyl transferases.
Many serine hydrolases catalyze perhydrolysis, the reversible formation of peracids from carboxylic acids and hydrogen peroxide. Recently, we showed that a single amino acid substitution in the alcohol binding pocket, L29P, in Pseudomonas fluorescens (SIK WI) aryl esterase (PFE) increased the specificity constant of PFE for peracetic acid formation >100-fold [Bernhardt et al. (2005) Angew. Chem., Int. Ed. 44, 2742]. In this paper, we extend this work to address the three following questions. First, what is the molecular basis of the increase in perhydrolysis activity? We previously proposed that the L29P substitution creates a hydrogen bond between the enzyme and hydrogen peroxide in the transition state. Here we report two X-ray structures of L29P PFE that support this proposal. Both structures show a main chain carbonyl oxygen closer to the active site serine as expected. One structure further shows acetate in the active site in an orientation consistent with reaction by an acyl-enzyme mechanism. We also detected an acyl-enzyme intermediate in the hydrolysis of epsilon-caprolactone by mass spectrometry. Second, can we further increase perhydrolysis activity? We discovered that the reverse reaction, hydrolysis of peracetic acid to acetic acid and hydrogen peroxide, occurs at nearly the diffusion limited rate. Since the reverse reaction cannot increase further, neither can the forward reaction. Consistent with this prediction, two variants with additional amino acid substitutions showed 2-fold higher k(cat), but K(m) also increased so the specificity constant, k(cat)/K(m), remained similar. Third, how does the L29P substitution change the esterase activity? Ester hydrolysis decreased for most esters (75-fold for ethyl acetate) but not for methyl esters. In contrast, L29P PFE catalyzed hydrolysis of epsilon-caprolactone five times more efficiently than wild-type PFE. Molecular modeling suggests that moving the carbonyl group closer to the active site blocks access for larger alcohol moieties but binds epsilon-caprolactone more tightly. These results are consistent with the natural function of perhydrolases being either hydrolysis of peroxycarboxylic acids or hydrolysis of lactones.
The structures of cofactor-free haloperoxidases from Streptomyces aureofaciens, Streptomyces lividans, and Pseudomonas fluorescens have been determined at resolutions between 1.9 A and 1.5 A. The structures of two enzymes complexed with benzoate or propionate identify the binding site for the organic acids which are required for the haloperoxidase activity. Based on these complexes and on the structure of an inactive variant, a reaction mechanism is proposed for the halogenation reaction with peroxoacid and hypohalous acid as reaction intermediates. Comparison of the structures suggests that a specific halide binding site is absent in the enzymes but that hydrophobic organic compounds may fit into the active site pocket for halogenation at preferential sites.
Acyl transfer is a key reaction in biosynthesis, including synthesis of antibiotics and polyesters. Although researchers have long recognized the similar protein fold and catalytic machinery in acyltransferases and hydrolases, the molecular basis for the different reactivity has been a long-standing mystery. By comparison of X-ray structures, we identified a different oxyanion-loop orientation in the active site. In esterases/lipases a carbonyl oxygen points toward the active site, whereas in acyltransferases a NH of the main-chain amide points toward the active site. Amino acid sequence comparisons alone cannot identify such a difference in the main-chain orientation. To identify how this difference might change the reaction mechanism, we solved the X-ray crystal structure of Pseudomonas fluorescens esterase containing a sulfonate transition-state analogue bound to the active-site serine. This structure mimics the transition state for the attack of water on the acyl-enzyme and shows a bridging water molecule between the carbonyl oxygen mentioned above and the sulfonyl oxygen that mimics the attacking water. A possible mechanistic role for this bridging water molecule is to position and activate the attacking water molecule in hydrolases, but to deactivate the attacking water molecule in acyl transferases.
Many serine hydrolases catalyze perhydrolysis, the reversible formation of peracids from carboxylic acids and hydrogen peroxide. Recently, we showed that a single amino acid substitution in the alcohol binding pocket, L29P, in Pseudomonas fluorescens (SIK WI) aryl esterase (PFE) increased the specificity constant of PFE for peracetic acid formation >100-fold [Bernhardt et al. (2005) Angew. Chem., Int. Ed. 44, 2742]. In this paper, we extend this work to address the three following questions. First, what is the molecular basis of the increase in perhydrolysis activity? We previously proposed that the L29P substitution creates a hydrogen bond between the enzyme and hydrogen peroxide in the transition state. Here we report two X-ray structures of L29P PFE that support this proposal. Both structures show a main chain carbonyl oxygen closer to the active site serine as expected. One structure further shows acetate in the active site in an orientation consistent with reaction by an acyl-enzyme mechanism. We also detected an acyl-enzyme intermediate in the hydrolysis of epsilon-caprolactone by mass spectrometry. Second, can we further increase perhydrolysis activity? We discovered that the reverse reaction, hydrolysis of peracetic acid to acetic acid and hydrogen peroxide, occurs at nearly the diffusion limited rate. Since the reverse reaction cannot increase further, neither can the forward reaction. Consistent with this prediction, two variants with additional amino acid substitutions showed 2-fold higher k(cat), but K(m) also increased so the specificity constant, k(cat)/K(m), remained similar. Third, how does the L29P substitution change the esterase activity? Ester hydrolysis decreased for most esters (75-fold for ethyl acetate) but not for methyl esters. In contrast, L29P PFE catalyzed hydrolysis of epsilon-caprolactone five times more efficiently than wild-type PFE. Molecular modeling suggests that moving the carbonyl group closer to the active site blocks access for larger alcohol moieties but binds epsilon-caprolactone more tightly. These results are consistent with the natural function of perhydrolases being either hydrolysis of peroxycarboxylic acids or hydrolysis of lactones.
        
Title: Stereoselective esterase from Pseudomonas putida IFO12996 reveals alpha/beta hydrolase folds for D-beta-acetylthioisobutyric acid synthesis Elmi F, Lee HT, Huang JY, Hsieh YC, Wang YL, Chen YJ, Shaw SY, Chen CJ Ref: Journal of Bacteriology, 187:8470, 2005 : PubMed
Esterase (EST) from Pseudomonas putida IFO12996 catalyzes the stereoselective hydrolysis of methyl dl-beta-acetylthioisobutyrate (dl-MATI) to produce d-beta-acetylthioisobutyric acid (DAT), serving as a key intermediate for the synthesis of angiotensin-converting enzyme inhibitors. The EST gene was cloned and expressed in Escherichia coli; the recombinant protein is a non-disulfide-linked homotrimer with a monomer molecular weight of 33,000 in both solution and crystalline states, indicating that these ESTs function as trimers. EST hydrolyzed dl-MATI to produce DAT with a degree of conversion of 49.5% and an enantiomeric excess value of 97.2% at an optimum pH of about 8 to 10 and an optimum temperature of about 57 to 67 degrees C. The crystal structure of EST has been determined by X-ray diffraction to a resolution of 1.6 A, confirming that EST is a member of the alpha/beta hydrolase fold superfamily of enzymes and includes a catalytic triad of Ser97, Asp227, and His256. The active site is located approximately in the middle of the molecule at the end of a pocket approximately 12 A deep. EST can hydrolyze the methyl ester group without affecting the acetylthiol ester moiety in dl-MATI. The examination of substrate specificity of EST toward other linear esters revealed that the enzyme showed specific activity toward methyl esters and that it recognized the configuration at C-2.
The structure of PFE, an aryl esterase from Pseudomonas fluorescens, has been solved to a resolution of 1.8 A by X-ray diffraction and shows a characteristic alpha/beta-hydrolase fold. In addition to catalyzing the hydrolysis of esters in vitro, PFE also shows low bromoperoxidase activity. PFE shows highest structural similarity, including the active-site environment, to a family of non-heme bacterial haloperoxidases, with an r.m.s. deviation in 271 C(alpha) atoms between PFE and its five closest structural neighbors averaging 0.8 A. PFE has far less similarity (r.m.s. deviation in 218 C(alpha) atoms of 5.0 A) to P. fluorescens carboxyl esterase. PFE favors activated esters with small acyl groups, such as phenyl acetate. The X-ray structure of PFE reveals a significantly occluded active site. In addition, several residues, including Trp28 and Met95, limit the size of the acyl-binding pocket, explaining its preference for small acyl groups.
        
Title: The crystal structure of a (-) gamma-lactamase from an Aureobacterium species reveals a tetrahedral intermediate in the active site Line K, Isupov MN, Littlechild JA Ref: Journal of Molecular Biology, 338:519, 2004 : PubMed
The structure of the recombinant (-) gamma-lactamase from an Aureobacterium species has been solved at 1.73A resolution in the cubic space group F23 with unit cell parameters a=b=c=240.6A. The trimeric enzyme has an alpha/beta hydrolase fold and closely resembles the cofactor free haloperoxidases. The structure has been solved in complex with a covalently bound ligand originating from the host cell and also in the unligated form. The associated density in the former structure has been interpreted as the two-ring ligand (3aR,7aS)-3a,4,7,7a-tetrahydro-benzo [1,3] dioxol-2-one which forms a tetrahedral complex with OG of the catalytic Ser98. Soaks of these crystals with the industrial substrate gamma-lactam or its structural analogue, norcamphor, result in the displacement of the ligand from the enzyme active site, thereby allowing determination of the unligated structure. The presence of the ligand in the active site protects the enzyme from serine hydrolase inhibitors. Cyclic ethylene carbonate, the first ring of the ligand, was shown to be a substrate of the enzyme.
The structures of cofactor-free haloperoxidases from Streptomyces aureofaciens, Streptomyces lividans, and Pseudomonas fluorescens have been determined at resolutions between 1.9 A and 1.5 A. The structures of two enzymes complexed with benzoate or propionate identify the binding site for the organic acids which are required for the haloperoxidase activity. Based on these complexes and on the structure of an inactive variant, a reaction mechanism is proposed for the halogenation reaction with peroxoacid and hypohalous acid as reaction intermediates. Comparison of the structures suggests that a specific halide binding site is absent in the enzymes but that hydrophobic organic compounds may fit into the active site pocket for halogenation at preferential sites.
        
Title: The metal-ion-free oxidoreductase from Streptomyces aureofaciens has an alpha/beta hydrolase fold Hecht HJ, Sobek H, Haag T, Pfeifer O, van Pee KH Ref: Nat Struct Biol, 1:532, 1994 : PubMed
The crystal structure of the bromoperoxidase A2 from Streptomyces aureofaciens (ATCC 10762) has been determined by isomorphous replacement and refined to 2.05 A resolution with an R-value of 18.4%. The enzyme catalyzes the bromination of organic compounds in the presence of bromide and peroxide. The structure confirms the absence of cofactors such as metal ions or haem groups and shows the general topology of the alpha/beta hydrolase fold. The active centre is at the end of a deep pocket and includes a catalytic triad of Ser 98, Asp 228 and His 257. The active centre is connected by a narrow tunnel to a second pocket on the enzyme surface.