The natural substrate of hydroxynitrile lyase from rubber tree (HbHNL, Hevea brasiliensis) is acetone cyanohydrin, but synthetic applications usually involve aromatic cyanohydrins such as mandelonitrile. To increase the activity of HbHNL toward this unnatural substrate, we replaced active site residues in HbHNL with the corresponding ones from esterase SABP2 (salicylic acid binding protein 2). Although this enzyme catalyzes a different reaction (hydrolysis of esters), its natural substrate (methyl salicylate) contains an aromatic ring. Three of the eleven single-amino-acid-substitution variants of HbHNL reacted more rapidly with mandelonitrile. The best was HbHNL-L121Y, with a k_cat 4.2 times higher and high enantioselectivity. Site-saturation mutagenesis at position 121 identified three other improved variants. We hypothesize that the smaller active site orients the aromatic substrate more productively.

Some serine hydrolases also catalyze a promiscuous reaction— reversible perhydrolysis of carboxylic acids to make peroxycarboxylic acids. Five X-ray crystal structures of these carboxylic acid perhydrolases show a proline in the oxyanion loop. Here, we test whether this proline is essential for high perhydrolysis activity using Pseudomonas fluorescens esterase (PFE). The L29P variant of this esterase catalyzes perhydrolysis 43-fold faster (k_cat comparison) than the wild type. Surprisingly, saturation mutagenesis at the 29 position of PFE identified six other amino acid substitutions that increase perhydrolysis of acetic acid at least fourfold over the wild type. The best variant, L29I PFE, catalyzed perhydrolysis 83-times faster (k_cat comparison) than wild-type PFE and twice as fast as L29P PFE. Despite the different amino acid in the oxyanion loop, L29I PFE shows a similar selectivity for hydrogen peroxide over water as L29P PFE (β₀=170 vs. 160 M⁻¹), and a similar fast formation of acetyl-enzyme (140 vs. 62 U mg⁻¹). X-ray crystal structures of L29I PFE with and without bound acetate show an unusual mixture of two different oxyanion loop conformations. The type II β-turn conformation resembles the wild-type structure and is unlikely to increase perhydrolysis, but the type I β-turn conformation creates a binding site for a second acetate. Modeling suggests that a previously proposed mechanism for L29P PFE can be extended to include L29I PFE, so that an acetate accepts a hydrogen bond to promote faster formation of the acetyl-enzyme.

Several serine hydrolases catalyze a promiscuous reaction: perhydrolysis of carboxylic acids to form peroxycarboxylic acids. The working hypothesis is that perhydrolases are more selective than esterases for hydrogen peroxide over water. In this study, we tested this hypothesis, and focused on L29P-PFE (Pseudomonas fluorescens esterase), which catalyzes perhydrolysis of acetic acid 43-fold faster than wild-type PFE. This hypothesis predicts that L29P-PFE should be approximately 43-fold more selective for hydrogen peroxide than wild-type PFE, but experiments show that L29P-PFE is less selective. The ratio of hydrolysis to perhydrolysis of methyl acetate at different concentrations of hydrogen peroxide fit a kinetic model for nucleophile selectivity. L29P-PFE (β₀=170 M⁻¹) is approximately half as selective for hydrogen peroxide over water than wild-type PFE (β₀=330 M⁻¹), which contradicts the working hypothesis. An alternative hypothesis is that carboxylic acid perhydrolases increase perhydrolysis by forming the acyl-enzyme intermediate faster. Consistent with this hypothesis, the rate of acetyl-enzyme formation, measured by ¹⁸O-water exchange into acetic acid, was 25-fold faster with L29P-PFE than with wild-type PFE, which is similar to the 43-fold faster perhydrolysis with L29P-PFE. Molecular modeling of the first tetrahedral intermediate (T_d1) suggests that a closer carbonyl group found in perhydrolases accepts a hydrogen bond from the leaving group water. This revised understanding can help design more efficient enzymes for perhydrolysis and shows how subtle changes can create new, unnatural functions in enzymes.

Lipase from Candida rugosa shows high enantioselectivity toward α-substituted chiral acids such as 2-arylpropionic acids. To understand how Candida rugosa lipase (CRL) distinguishes between enantiomers of chiral acids, we determined the X-ray crystal structure of a transition-state analog covalently linked to CRL. CRL shows moderate enantioselectivity (E=23) toward methyl 2-methoxy-2-phenylacetate, 1-methyl ester, favoring the (S)-enantiomer. We synthesized phosphonate (R_C,R_PS_P)-3, which, upon reaction with CRL, mimics the transition state for hydrolysis of (S)-1-methyl ester, the fast-reacting enantiomer. An X-ray crystal structure of this complex shows a catalytically productive orientation with the phenyl ring in the hydrophobic tunnel of the lipase. Phe345 crowds the region near the substrate stereocenter. Computer modeling of the slow-reacting enantiomer examined four possible conformations for the corresponding slow-reacting enantiomer: three conformations where two substituents at the stereocenter have been exchanged relative to the fast-reacting enantiomer and one conformation with an umbrella-like inversion orientation. Each of these orientations disrupts the orientation of the catalytic histidine, but the molecular basis for disruption differs in each case showing that multiple mechanisms are required for high enantioselectivity.

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.

One useful synthetic reaction missing from nature's toolbox is the direct hydrogenation of substrates using hydrogen. Instead nature uses cofactors like NADH to reduce organic substrates, which adds complexity and cost to these reductions. To create an enzyme that can directly reduce organic substrates with hydrogen, researchers have combined metal hydrogenation catalysts with proteins. One approach is an indirect link where a ligand is linked to a protein and the metal binds to the ligand. Another approach is direct linking of the metal to protein, but nonspecific binding of the metal limits this approach. Herein, we report a direct hydrogenation of olefins catalyzed by rhodium(I) bound to carbonic anhydrase (CA-[Rh]). We minimized nonspecific binding of rhodium by replacing histidine residues on the protein surface using site-directed mutagenesis or by chemically modifying the histidine residues. Hydrogenation catalyzed by CA-[Rh] is slightly slower than for uncomplexed rhodium(I), but the protein environment induces stereoselectivity favoring cis- over trans-stilbene by about 20:1. This enzyme is the first cofactor-independent reductase that reduces organic molecules using hydrogen. This catalyst is a good starting point to create variants with tailored reactivity and selectivity. This strategy to insert transition metals in the active site of metalloenzymes opens opportunities to a wider range of enzyme-catalyzed reactions.

Candida antarctica lipase B catalyzed the stereoselective ammoniolysis of N-alkyl aziridine-2-carboxylates in tBuOH saturated with ammonia and yielded the (2S)-aziridine-2-carboxamide and unreacted (2R)-aziridine-2-carboxylate. Varying the N-1 substituent on the aziridine ring changed the rate and stereoselectivity of the reaction. Substrates with a benzyl substituent or a (1′R)-1-phenylethyl substituent reacted approximately ten times faster than substrates with a (1′S)-1-phenylethyl substituent. Substrates with a benzyl substituent showed little stereoselectivity (E=5–7) while substrates with either a (1′R)- or (1′S)-1-phenylethyl substituent showed high stereoselectivity (D>50). Molecular modeling by using the current paradigm for enantioselectivity—binding of the slow enantiomer by an exchange-of-substituents orientation—could not account for the experimental results. However, modeling an umbrella-like-inversion orientation for the slow enantiomer could account for the experimental results. Steric hindrance between the methyl in the (1′S)-1-phenylethyl substituent and Thr138 and Ile189 in the acyl-binding site likely accounts for the slow reaction. Enantioselectivity likely stems from an unfavorable interaction of the methine hydrogen with Thr40 for the slow enantiomer and from subtle differences in the orientations of the other three substituents. This success in rationalizing the enantioselectivity supports the notion that an umbrella-like-inversion orientation can contribute to enantioselectivity in lipases.

Lipases show high enantioselectivity toward a wide range of secondary alcohols. An empirical rule based on the relative sizes of the substituents predicts which enantiomer reacts faster. X-ray structures of lipases provide a molecular basis for this empirical rule: their alcohol-binding pocket contains large hydrophobic pocket open to solvent and another smaller pocket. This predictable enantiopreference of lipases allows the determination of the absolute configuration of secondary alcohols using lipase-catalyzed kinetic resolution. Researchers have used this relative method to determine the configuration of ∼50 secondary alcohols either as the only method or in combination with other methods. Chirality, 2008. © 2008 Wiley-Liss, Inc.

One often-cited weakness of biocatalysis is the lack of mirror-image enzymes for the formation of either enantiomer of a product in asymmetric synthesis. Enantiocomplementary enzymes exist as the solution to this problem in nature. These enzyme pairs, which catalyze the same reaction but favor opposite enantiomers, are not mirror-image molecules; however, they contain active sites that are functionally mirror images of one another. To create mirror-image active sites, nature can change the location of the binding site and/or the location of key catalytic groups. In this Minireview, X-ray crystal structures of enantiocomplementary enzymes are surveyed and classified into four groups according to how the mirror-image active sites are formed.