Mutation esterases

Esterases have a catalytic function and mechanism similar to those of lipases, but some structural aspects and the nature of substrates differ [4]. One can expect that the lessons learned from the directed evolution of lipases also apply to esterases. However, few efforts have been made in the directed evolution of enantioselective esterases, although previous work by Arnold had shown that the activity of esterases as catalysts in the hydrolysis of achiral esters can be enhanced [49]. An example regarding enantioselectivity involves the hydrolytic kinetic resolution of racemic esters catalyzed by Pseudomonasfluorescens esterase (PFE) [50]. Using a mutator strain and by screening very small libraries, low improvement in enantioselectivity was... 

Epothilones are a class of molecules that show anticancer activity. Production of a synthetic intermediate was investigated through the action of an esterase on various sterically hindered 3-hydroxy esters [76]. No initial activity was observed, so a Pseudomonasfluorescens esterase was transformed into a mutator strain Epicurian coli and screened using an indicator in the growth plates that would produce a red color if hydrolysis occurred. An ee of 25% was achieved from a variant containing two mutations. 

In a more detailed study, the same esterase P. fluorescens) was again subjected to mutagenesis using the same mutator strain, but also by saturation mutagenesis at selected positions 133a). In addition to 3-phenylbutyric acid ethyl ester (27), 3-bromo-2-methyl-propionic acid methyl ester rac-31) was chosen for the hydrolytic kinetic resolution, with the WT PFE showing an E factor of 12 in favor of the (5)-32. 

Directed evolution involves multiple rounds of random mutation and selection combined with gene shuffling to evolve enzymes towards desired properties (reviewed in Arnold and Moore, 1997 Kuchner and Arnold, 1997). The group of Arnold has succeeded in evolving a dimethylformamide (DMF)-sensitive esterase for the cleavage of the loracarbef-/>-nitrobenzyl ester into an esterase that remains active in 15% DW (Moore et al, 1997). Most of the mutations that had been found in the solvent-resistant mutants could not have been predicted using current computational methods. 

Another important problem is the development of insects resistant to insecticides. This often arises as a result of increased levels of carboxylesterases which hydrolyze both organophosphates and car-baryl.h/1 A mutation that changed a single active site glycine to aspartate in a carboxylesterase of a blowfly changed the esterase to an organophosphorus hydrolase which protected the fly against insecticides.)... 

Fig. 7. Molecular model of the pNB esterase showing positions of antibiotic p-nitrobenzyl ester substrate (white CPK structure), catalytic residues (S189, E310, and H399), and beneficial mutations accumulated during directed evolution. Mutations at positions 322 and 370 are believed to improve expression, while the remaining six substitutions improve specific activity [2]. Arrows indicate the position of new mutations found after DNA shuffling...

The positions of the amino acid substitutions identified in the various pNB esterases are illustrated in Fig. 7, on a model of the pNB esterase developed from the X-ray crystal structures of homologous enzymes [2]. Known beneficial mutations are indicated also shown are the mutations believed to be neutral or to affect expression. Positions of the additional translated mutations found in the variants produced by DNA shuffling are indicated by the white arrows. None of the effective amino acid substitutions lie in segments of the esterase predicted to interact directly with the bound substrate. It may be that the amino acid substitutions sampled at positions adjacent to the substrate were all... 

These investigations also showed that the conversion of ECB to ECB nucleus would proceed more rapidly if ECB were first solubilized in a suitable solvent such as methanol or acetone. However, if the concentration of solvent was too high, the enzyme activity was reduced. Ideally, the enzyme itself could be tailored to suit the industrially preferred conditions (e.g., to make it more resistant to solvent or active at a different pH). One method for achieving this is to use directed evolution [42], whereby genes encoding the enzyme are mutated, screened and then recombined in vitro. Although the contributions of individual amino acid mutations are small, the accumulation of multiple mutations by directed evolution allows significant improvement in the biocatalyst for reactions on substrates or under conditions not already optimized in nature. This approach was used by Arnold and Moore [43] to make a 150-fold improvement in the activity of a -nitrobenzyl esterase in the presence of 15% DMSO. 

LiCata and Ackers report that mutations that are not directly in contact can be non-additive (1995). They find that most mutations exhibit some degree of non-additivity that cannot be explained by short-range disruptions. A structural study of mutants of pNB esterase generated by directed evolution supports this observation (Spiller et al., 1999 see chapter by Orencia, Hanson, and Stevens in this volume). In this case, the influence of a mutation was realized through small backbone shifts, spatially distant from the mutated residue. Non-additivity can result when these perturbed regions overlap (Skinner and Terwilliger, 1996). 

Fig. 8. MolScript (Kraulis, 1991) diagram of the three-dimensional structure of />NB esterase variant 8G8 (Spiller et al., 1999). Mutated residues are shown in black ball-and-stick. Catalytic residues are shown in white ball-and stick. Black portions indicate stabilized loop regions.

The plot of the stabilities and activities of clones from the first generation S41 random mutant library shows once again that most mutations are detrimental to stability and activity (Fig. 14). However, compared to the esterase library (Fig. 7), there are more mutants with improvements in both properties, suggesting that the two enzymes have different adaptive potentials. This may be due to the relatively poor stability of S41, or it may reflect constraints intrinsic to the three-dimensional structures of the two proteins. Evidence for the former can be found by comparing the results for the first generations of the psychrophilic sub-tilisin S41 and the mesophilic subtilisin E. Screening 864 mutants of S41 yielded nine thermostabilized variants (a hit rate of approximately 1%) (Miyazaki and Arnold, 1999) in contrast, screening 5000 subtilisin E mutants identified five thermostable variants (a hit rate of only 0.1%) (Zhao and Arnold, 1999). 

Characteristics of Thermostabilizing Mutations in pNB Esterase, Subtilisin E, and Subtilisin S41... 

G412E surface, helix 13 5 Forms salt bridge with Arg415 in 414-420 loop (which is unstructured in wild-type esterase) reinforcing I60V mutation. 

The most thermophilic variant of j/NB esterase, 8G8, has only thirteen mutations compared to the wild-type esterase, making it 97% identical to the wild-type esterase sequence, with a root-mean-square deviation of only 0.44 A between the two C backbone structures. As with the 5-6C8 organophile structure, the catalytic triads of 8G8 and wild-type / NB esterase are superimposable. This high sequence and structural identity, in conjunction with the availability of crystal structures for both the wild type and thermophile, affords an interesting opportunity to study the structural basis for thermostability. Thermophile 8G8 is the product of eight generations of directed evolution, screening for retention of activity... 

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