Hydroxynitrile lyases (HNL's) belonging to the alpha/beta-hydrolase-fold superfamily evolved from esterases approximately 100 million years ago. Reconstruction of an ancestral hydroxynitrile lyase in the alpha/beta-hydrolase fold superfamily yielded a catalytically active hydroxynitrile lyase, HNL1. Several properties of HNL1 differ from the modern HNL from rubber tree (HbHNL). HNL1 favors larger substrates as compared to HbHNL, is two-fold more catalytically promiscuous for ester hydrolysis (p-nitrophenyl acetate) as compared to mandelonitrile cleavage, and resists irreversible heat inactivation to 35 degrees C higher than for HbHNL. We hypothesized that the x-ray crystal structure of HNL1 may reveal the molecular basis for the differences in these properties. The x-ray crystal structure solved to 1.96-A resolution shows the expected alpha/beta-hydrolase fold, but a 60% larger active site as compared to HbHNL. This larger active site echoes its evolution from esterases since related esterase SABP2 from tobacco also has a 38% larger active site than HbHNL. The larger active site in HNL1 likely accounts for its ability to accept larger hydroxynitrile substrates. Site-directed mutagenesis of HbHNL to expand the active site increased its promiscuous esterase activity 50-fold, consistent with the larger active site in HNL1 being the primary cause of its promiscuous esterase activity. Urea-induced unfolding of HNL1 indicates that it unfolds less completely than HbHNL (m-value = 0.63 for HNL1 vs 0.93 kcal/mol.M for HbHNL), which may account for the ability of HNL1 to better resist irreversible inactivation upon heating. The structure of HNL1 shows changes in hydrogen bond networks that may stabilize regions of the folded structure.
Title: Identical active sites in hydroxynitrile lyases show opposite enantioselectivity and reveal possible ancestral mechanism Jones BJ, Bata Z, Kazlauskas RJ Ref: ACS Catal, 7:4221, 2017 : PubMed
Evolutionarily related hydroxynitrile lyases from rubber tree (HbHNL) and from Arabidopsis thaliana (AtHNL) follow different catalytic mechanisms with opposite enantioselectivity toward mandelonitrile. We hypothesized that the HbHNL-like mechanism evolved from an enzyme with an AtHNL-like mechanism. We created ancestor-like composite active-sites in each scaffold to elucidate how this transition may have occurred. Surprisingly, a composite active site in HbHNL maintained (S)-selectivity, while the identical set of active site residues in AtHNL maintained (R)-selectivity. Composite active-site mutants that are (S)-selective without the Lys236 and Thr11 that are required for the classical (S)-HNL mechanism suggests a new mechanism. Modeling suggested a possibility for this new mechanism that does not exist in modern enzymes. Thus, the last common ancestor of HbHNL and AtHNL may have used an extinct mechanism, not the AtHNL-like mechanism. Multiple mechanisms are possible with the same catalytic residues and residues outside the active site strongly influence mechanism and enantioselectivity.
Title: Comparison of Five Protein Engineering Strategies for Stabilizing an alpha/beta-Hydrolase Jones BJ, Lim HY, Huang J, Kazlauskas RJ Ref: Biochemistry, 56:6521, 2017 : PubMed
A review of the previous stabilization of alpha/beta-hydrolase fold enzymes revealed many different strategies, but no comparison of strategies on the same enzyme. For this reason, we compared five strategies to identify stabilizing mutations in a model alpha/beta-hydrolase fold enzyme, salicylic acid binding protein 2, to reversible denaturation by urea and to irreversible denaturation by heat. The five strategies included one location agnostic approach (random mutagenesis using error-prone polymerase chain reaction), two structure-based approaches [computational design (Rosetta, FoldX) and mutation of flexible regions], and two sequence-based approaches (addition of proline at locations where a more stable homologue has proline and mutation to consensus). All strategies identified stabilizing mutations, but the best balance of success rate, degree of stabilization, and ease of implementation was mutation to consensus. A web-based automated program that predicts substitutions needed to mutate to consensus is available at http://kazlab.umn.edu .
Catalytic promiscuity is a useful, but accidental, enzyme property, so finding catalytically promiscuous enzymes in nature is inefficient. Some ancestral enzymes were branch points in the evolution of new enzymes and are hypothesized to have been promiscuous. To test the hypothesis that ancestral enzymes were more promiscuous than their modern descendants, we reconstructed ancestral enzymes at four branch points in the divergence hydroxynitrile lyases (HNL's) from esterases approximately 100 million years ago. Both enzyme types are alpha/beta-hydrolase-fold enzymes and have the same catalytic triad, but differ in reaction type and mechanism. Esterases catalyze hydrolysis via an acyl enzyme intermediate, while lyases catalyze an elimination without an intermediate. Screening ancestral enzymes and their modern descendants with six esterase substrates and six lyase substrates found higher catalytic promiscuity among the ancestral enzymes (P < 0.01). Ancestral esterases were more likely to catalyze a lyase reaction than modern esterases, and the ancestral HNL was more likely to catalyze ester hydrolysis than modern HNL's. One ancestral enzyme (HNL1) along the path from esterase to hydroxynitrile lyases was especially promiscuous and catalyzed both hydrolysis and lyase reactions with many substrates. A broader screen tested mechanistically related reactions that were not selected for by evolution: decarboxylation, Michael addition, gamma-lactam hydrolysis and 1,5-diketone hydrolysis. The ancestral enzymes were more promiscuous than their modern descendants (P = 0.04). Thus, these reconstructed ancestral enzymes are catalytically promiscuous, but HNL1 is especially so.
Title: Stabilization of an alpha/beta-Hydrolase by Introducing Proline Residues: Salicylic Acid Binding Protein 2 from Tobacco Huang J, Jones BJ, Kazlauskas RJ Ref: Biochemistry, 54:4330, 2015 : PubMed
