Switching catalysis from hydrolysis to perhydrolysis in Pseudomonas fluorescens esterase

Abstract

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.

Figure 1

Proposed mechanisms for perhydrolysis of acetic acid. The numbering corresponds to the active site of PFE. a) The ping-pong bi-bi mechanism involves an acetyl-enzyme intermediate. The first diagram shows the enzyme-acetic acid complex. The γ-hydroxyl group of active site serine 94 is a nucleophile that attacks the carbonyl group of acetic acid to form a tetrahedral intermediate (not shown) via the black curved arrows. Next, this tetrahedral intermediate collapses via the release of water (gray curved arrows) to form an acetyl-enzyme intermediate. Finally hydrogen peroxide binds to yield the complex shown in the second diagram. The N–H's of M95 and W28, called the oxyanion hole, donate hydrogen bonds to the carbonyl oxygen. Nucleophilic attack of hydrogen peroxide on the acyl enzyme forms a second tetrahedral intermediate. In wild-type PFE, the carbonyl oxygen of W28 is too far from the hydrogen peroxide to form a hydrogen bond. The L29P substitution moves this carbonyl 0.5 Å closer to the catalytic serine side-chain allowing a hydrogen bond to form and stabilize the tetrahedral intermediate. b) The ordered bi-bi noncovalent mechanism proposed by Bugg (11) does not form an acyl-enzyme intermediate. Both acetic acid and hydrogen peroxide bind the enzyme simultaneously. In this mechanism, acetic acid does not bind to the oxyanion hole, but the γ-hydroxyl group of active site S94 and N–H of W28 donate a hydrogen bond to the carbonyl oxygen of acetic acid. Hydrogen peroxide attacks the bound acetic acid to form a tetrahedral intermediate without covalent links to the enzyme.

Figure 2

Active site x-ray crystal structures of L29P PFE and L29P PFE/acetate. A) Superposition of L29P PFE (green CPK colors, PDB ID: 3hea) and wild-type PFE (white CPK colors, PDB ID: 1va4) show similar conformations of the catalytic triad residues. (Only H251 and S94 are shown.) The leucine 29 to proline substitution changes the W28 to L29 peptide bond conformation from trans to cis thereby shifting the main chain carbonyl group of W28 closer to the active site serine by 1.1 Å. A second difference is a shift in the indole ring of the W28 residue by ∼1.3 Å for the 7 position. B) Superposition of the structures of L29P PFE/acetate (green CPK colors, PDB ID: 3hi4) and perhydrolase CPO-F soaked with propionate (magenta CPK colors, PDB ID: 1a8s). Both show a similar orientations of the W28 carbonyl group and indole ring, but the acetate, propanoate and active site water molecules (WAT1, orange for L29P, magenta for CPO-F) have different orientations. C) L29P PFE/acetate (PDB ID: 3hi4) shows the substrate acetate in an orientation consistent with formation of an acetyl-enzyme intermediate. The acetate carbonyl oxygen accept hydrogen bonds from the two amide N-H's that form the oxyanion hole (M95, W28). The carbonyl carbon of acetate is 2.8 Å from S94-Oγ in this monomer and ranges from 2.8 to 3.1 Å in the six monomers in the asymmetric unit.

Figure 2

Active site x-ray crystal structures of L29P PFE and L29P PFE/acetate. A) Superposition of L29P PFE (green CPK colors, PDB ID: 3hea) and wild-type PFE (white CPK colors, PDB ID: 1va4) show similar conformations of the catalytic triad residues. (Only H251 and S94 are shown.) The leucine 29 to proline substitution changes the W28 to L29 peptide bond conformation from trans to cis thereby shifting the main chain carbonyl group of W28 closer to the active site serine by 1.1 Å. A second difference is a shift in the indole ring of the W28 residue by ∼1.3 Å for the 7 position. B) Superposition of the structures of L29P PFE/acetate (green CPK colors, PDB ID: 3hi4) and perhydrolase CPO-F soaked with propionate (magenta CPK colors, PDB ID: 1a8s). Both show a similar orientations of the W28 carbonyl group and indole ring, but the acetate, propanoate and active site water molecules (WAT1, orange for L29P, magenta for CPO-F) have different orientations. C) L29P PFE/acetate (PDB ID: 3hi4) shows the substrate acetate in an orientation consistent with formation of an acetyl-enzyme intermediate. The acetate carbonyl oxygen accept hydrogen bonds from the two amide N-H's that form the oxyanion hole (M95, W28). The carbonyl carbon of acetate is 2.8 Å from S94-Oγ in this monomer and ranges from 2.8 to 3.1 Å in the six monomers in the asymmetric unit.

Figure 2

Active site x-ray crystal structures of L29P PFE and L29P PFE/acetate. A) Superposition of L29P PFE (green CPK colors, PDB ID: 3hea) and wild-type PFE (white CPK colors, PDB ID: 1va4) show similar conformations of the catalytic triad residues. (Only H251 and S94 are shown.) The leucine 29 to proline substitution changes the W28 to L29 peptide bond conformation from trans to cis thereby shifting the main chain carbonyl group of W28 closer to the active site serine by 1.1 Å. A second difference is a shift in the indole ring of the W28 residue by ∼1.3 Å for the 7 position. B) Superposition of the structures of L29P PFE/acetate (green CPK colors, PDB ID: 3hi4) and perhydrolase CPO-F soaked with propionate (magenta CPK colors, PDB ID: 1a8s). Both show a similar orientations of the W28 carbonyl group and indole ring, but the acetate, propanoate and active site water molecules (WAT1, orange for L29P, magenta for CPO-F) have different orientations. C) L29P PFE/acetate (PDB ID: 3hi4) shows the substrate acetate in an orientation consistent with formation of an acetyl-enzyme intermediate. The acetate carbonyl oxygen accept hydrogen bonds from the two amide N-H's that form the oxyanion hole (M95, W28). The carbonyl carbon of acetate is 2.8 Å from S94-Oγ in this monomer and ranges from 2.8 to 3.1 Å in the six monomers in the asymmetric unit.

Figure 3

Deconvoluted electrospray-ionization spectra show an acyl-enzyme intermediate during the PFE-L29P-catalyzed hydrolysis of ε-caprolactone in citrate/formate buffer pH ∼5.5. Without substrate shows the major peak at 30,912 Da, which is consistent with the calculated value of 30,911.9 Da using ProtParam (15). Addition of 25 mM ε-caprolactone shows an additional peak at 31,026 Da, which is 114 Da higher that the free enzyme. This peak is assigned as the covalent acyl-enzyme intermediate. Peaks at lower mass are present in both spectra and are not identified.

Figure 4

Mesh showing water-accessible regions in the active sites of wild-type PFE and L29P with the modeled tetrahedral intermediate for the acetylation of active site serine by ethyl acetate (T_d1). (A) The alcohol pocket accommodates the ethyl group of the tetrahedral intermediate and could also accept a longer alcohol, but not larger acyl groups. The acyl pocket of wild-type PFE is small while the alcohol pocket is larger, as shown on the diagram on the right side. (B) The leucine to proline substitution shifts the main chain W28 carbonyl which pinches off the alcohol pocket. The ethyl group is forced to adopt an unfavorable conformation in the enlarged acyl pocket caused by a shift in the indole ring of W28. A diagram on the right shows that the acyl pocket of L29P is larger than the alcohol pocket. The mesh shows regions accessible to a sphere with a radius of 1.4 Å, which models a water molecule. Water molecules were removed before modeling the mesh regions.

Figure 5