Catalytic Promiscuity of Ancestral Esterases and Hydroxynitrile Lyases

Sun

et al. 2016

Appl Environ Microbiol

Promiscuous enzymes are generally considered to be starting points in the evolution of offspring enzymes with more specific or even novel catalytic activities, which is the molecular basis of producing new biological functions. Mhg, a typical ␣/␤ fold hydrolase, was previously reported to have both ␥-lactamase and perhydrolase activities. However, despite having high structural similarity to and sharing an identical catalytic triad with an extensively studied esterase from Pseudomonas fluorescens, this enzyme did not show any esterase activity. Molecular docking and sequence analysis suggested a possible role for the entry of the binding pocket in blocking the entrance tunnel, preventing the ester compounds from entering into the pocket. By engineering the entrance tunnel with only one or two amino acid substitutions, we successfully obtained five esterase variants of Mhg. The variants exhibited a very broad substrate acceptance, hydrolyzing not only the classical p-nitrophenol esters but also various types of chiral esters, which are widely used as drug intermediates. Site 233 at the entrance tunnel of Mhg was found to play a pivotal role in modulating the three catalytic activities by adjusting the size and shape of the tunnel, with different amino acid substitutions at this site facilitating different activities. Remarkably, the variant with the L233G mutation was a very specific esterase without any ␥-lactamase and perhydrolase activities. Considering the amino acid conservation and differentiation, this site could be a key target for future protein engineering. In addition, we demonstrate that engineering the entrance tunnel is an efficient strategy to regulate enzyme catalytic capabilities. IMPORTANCEPromiscuous enzymes can act as starting points in the evolution of novel catalytic activities, thus providing a molecular basis for the production of new biological functions. In this study, we identified a critical amino acid residue (Leu233) at the entry of the substrate tunnel of a promiscuous enzyme, Mhg. We found that substitution of this residue with smaller amino acids such as Gly, Ala, Ser, or Pro endowed the enzyme with novel esterase activity. Different amino acids at this site can facilitate different catalytic activities. These findings exhibited universal significance in this subset of ␣/␤ fold hydrolases, including Mhg. Furthermore, we demonstrate that engineering the entrance tunnel is an efficient strategy to evolve new enzyme catalytic capabilities. Our study has important implications for the regulation of enzyme catalytic promiscuity and development of protein engineering methodologies. C atalytic promiscuity refers to the ability of an enzyme to catalyze different substrates and different types of chemical reactions in the same active center. Although high substrate specificity has been well known, increasing evidence indicates that enzyme catalytic promiscuity exists widely in nature (1, 2, 3, 4). Recently, enzyme promiscuity has captured much attention because it is related to the...

Section: Discussionmentioning

confidence: 65%

Facilitating the Evolution of Esterase Activity from a Promiscuous Enzyme (Mhg) with Catalytic Functions of Amide Hydrolysis and Carboxylic Acid Perhydrolysis by Engineering the Substrate Entrance Tunnel

Yan

Sun

et al. 2016

Appl Environ Microbiol

“…Enzyme catalytic promiscuity is the hidden ability of an enzyme to catalyze more than one type of chemical transformation (1,2). Lipase is one of the most popular biocatalysts in this research area due to its excellent catalytic performance in many kinds of reactions (Aldol reaction, Morita-Baylis-Hillman reaction, Mannich reaction, Henry reaction, Markovnikov addition, Michael addition, Hantzsch reaction, etc.)…”

Section: Introductionmentioning

confidence: 99%

A mild and efficient Dakin reaction mediated by lipase

Chen

Green Chemistry Letters and Reviews

et al. 2017

We have developed a novel lipase-mediated method to realize the Dakin reaction. A wide range of hydroxylated benzaldehydes could be oxidized with high yields (from 90% to 97%) under mild reaction conditions. Moreover, this lipase-mediated reaction could be scaled up easily and Novozym 435 could be reused more than 10 runs without an obvious decrease in enzyme activity. This protocol expands the application of lipase in organic synthesis and offers a complementary route for the Dakin reaction.

“…Several of these ancestors were catalytically promiscuous 8,17 . HNL1 catalyzed mainly hydroxynitrile lyase cleavage, but also showed low remaining esterase activity.…”

Section: Introductionmentioning

confidence: 99%

Identical Active Sites in Hydroxynitrile Lyases Show Opposite Enantioselectivity and Reveal Possible Ancestral Mechanism

2017

Self Cite

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.