Structure‐guided consensus approach to create a more thermostable penicillin G acylase

Polizzi, Karen M.; Chaparro‐Riggers, Javier; Vazquez-Figueroa, Eduardo; Bommarius, Andreas S.

doi:10.1002/biot.200600029

Cited by 76 publications

(65 citation statements)

References 38 publications

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“…Consensus mutations comprise the most easily identifiable potential of stabilizing mutations that can boost protein evolvability (25). As less than half of the predicted consensus mutations actually have a stabilizing effect (24,36), and different mutations become compensatory at different evolutionary stages (as seen in R1-R2 versus R7 and later), spiking these mutations into libraries is a more effective strategy than the use of a prestabilized starting point (20).…”

Section: Discussionmentioning

confidence: 99%

Bridging the gaps in design methodologies by evolutionary optimization of the stability and proficiency of designed Kemp eliminase KE59

Khersonsky

Kiss

Röthlisberger

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

219

221

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Computational design is a test of our understanding of enzyme catalysis and a means of engineering novel, tailor-made enzymes. While the de novo computational design of catalytically efficient enzymes remains a challenge, designed enzymes may comprise unique starting points for further optimization by directed evolution. Directed evolution of two computationally designed Kemp eliminases, KE07 and KE70, led to low to moderately efficient enzymes (k cat ∕K m values of ≤5 × 10 4 M −1 s −1 ). Here we describe the optimization of a third design, KE59. Although KE59 was the most catalytically efficient Kemp eliminase from this design series (by k cat ∕K m , and by catalyzing the elimination of nonactivated benzisoxazoles), its impaired stability prevented its evolutionary optimization. To boost KE59's evolvability, stabilizing consensus mutations were included in the libraries throughout the directed evolution process. The libraries were also screened with less activated substrates. Sixteen rounds of mutation and selection led to >2,000-fold increase in catalytic efficiency, mainly via higher k cat values. The best KE59 variants exhibited k cat ∕K m values up to 0.6 × 10 6 M −1 s −1 , and k cat ∕k uncat values of ≤10 7 almost regardless of substrate reactivity. Biochemical, structural, and molecular dynamics (MD) simulation studies provided insights regarding the optimization of KE59. Overall, the directed evolution of three different designed Kemp eliminases, KE07, KE70, and KE59, demonstrates that computational designs are highly evolvable and can be optimized to high catalytic efficiencies.computational protein design | enzyme mimic T he endeavor of making enzyme-like catalysts spans several decades (1) with the Kemp elimination being a thoroughly explored model (2-7). In this activated model system, basecatalyzed proton elimination from carbon is concerted with the cleavage of nitrogen-oxygen bond, thus leading to the cyanophenol product (Fig. 1A). Activation of a carboxylate base catalyst by desolvation is efficiently mimicked by aprotic dipolar solvents such as acetonitrile and by various enzyme mimics. Effective alignment of the substrate and charge-dispersing interactions that stabilize the negatively charged transition state (TS) have also been achieved by various enzyme mimics that catalyze the Kemp elimination (8, 9). However, generation of a (wo)manmade active site that exhibits all these features and performs as well as natural enzymes, remains a challenge.Computational methods for predicting structure from sequence at atomic accuracy provide a new approach to enzyme engineering (10-12). The design involves two steps: (i) designing an active-site configuration that may confer efficient catalysis, (ii) computing a sequence that confers the desired configuration. Both steps are currently far from optimal, as the catalytic efficiency of designed enzymes falls far behind that of natural enzymes. Further, as with other enzyme mimics, computational designs tackle activated model systems and fail to catalyze cha...

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Section: Discussionmentioning

confidence: 99%

Bridging the gaps in design methodologies by evolutionary optimization of the stability and proficiency of designed Kemp eliminase KE59

Khersonsky

Kiss

Röthlisberger

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

219

221

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“…The high functionality of the consensus library can be attributed to the choice of specific point mutations and the application of eliminating criteria to reduce destructive mutations. It was previously shown that the datadriven consensus approach is useful in creating mutant libraries of high functionality, which enables the screening effort to be minimized (39,42,47,61). Low functionality of a random mutagenesis library may be a result of a high mutation rate and multiple alterations that can lead to destabilization and unfolding of the enzyme (36,66).…”

Section: Figmentioning

confidence: 99%

“…Protein engineering by introduction of ancestral/consensus residues to the enzyme protein sequence has been successfully used to evolve thermostable enzymes (39,(42)(43)(44)(45)(46). The underlying assumption of this approach is that conserved amino acids can contribute to the enzyme stability under extreme conditions (36,43,(46)(47)(48).…”

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confidence: 99%

Protein Engineering by Random Mutagenesis and Structure-Guided Consensus of Geobacillus stearothermophilus Lipase T6 for Enhanced Stability in Methanol

Dror

Shemesh

Dayan

et al. 2014

Appl Environ Microbiol

116

107

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The abilities of enzymes to catalyze reactions in nonnatural environments of organic solvents have opened new opportunities for enzyme-based industrial processes. However, the main drawback of such processes is that most enzymes have a limited stability in polar organic solvents. In this study, we employed protein engineering methods to generate a lipase for enhanced stability in methanol, which is important for biodiesel production. Two protein engineering approaches, random mutagenesis (error-prone PCR) and structure-guided consensus, were applied in parallel on an unexplored lipase gene from Geobacillus stearothermophilus T6. A high-throughput colorimetric screening assay was used to evaluate lipase activity after an incubation period in high methanol concentrations. Both protein engineering approaches were successful in producing variants with elevated half-life values in 70% methanol. The best variant of the random mutagenesis library, Q185L, exhibited 23-fold-improved stability, yet its methanolysis activity was decreased by one-half compared to the wild type. The best variant from the consensus library, H86Y/ A269T, exhibited 66-fold-improved stability in methanol along with elevated thermostability (؉4.3°C) and a 2-fold-higher fatty acid methyl ester yield from soybean oil. Based on in silico modeling, we suggest that the Q185L substitution facilitates a closed lid conformation that limits access for both the methanol and substrate excess into the active site. The enhanced stability of H86Y/A269T was a result of formation of new hydrogen bonds. These improved characteristics make this variant a potential biocatalyst for biodiesel production.

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“…Of the rational design methods, a structureguided consensus approach combines information about the quaternary structure with knowledge of a few sequences that have moderate identity to reduce the number of target residues in a given protein to be mutated. At present, it is considered an effi-cient and reliable method for improving enzymatic properties (16,(22)(23)(24)(25).…”

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confidence: 99%

Improving the Thermostability and Catalytic Efficiency of Bacillus deramificans Pullulanase by Site-Directed Mutagenesis

Duan

Chen

2013

Appl Environ Microbiol

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b Pullulanase (EC 3.2.1.41) is a well-known starch-debranching enzyme. Its instability and low catalytic efficiency are the major factors preventing its widespread application. To address these issues, Asp437 and Asp503 of the pullulanase from Bacillus deramificans were selected in this study as targets for site-directed mutagenesis based on a structure-guided consensus approach. Four mutants (carrying the mutations D503F, D437H, D503Y, and D437H/D503Y) were generated and characterized in detail. The results showed that the D503F, D437H, and D503Y mutants had an optimum temperature of 55°C and a pH optimum of 4.5, similar to that of the wild-type enzyme. However, the half-lives of the mutants at 60°C were twice as long as that of the wild-type enzyme. In addition, the D437H/D503Y double mutant displayed a larger shift in thermostability, with an optimal temperature of 60°C and a half-life at 60°C of more than 4.3-fold that of the wild-type enzyme. Kinetic studies showed that the K m values for the D503F, D437H, D503Y, and D437H/D503Y mutants decreased by 7.1%, 11.4%, 41.4%, and 45.7% and the K cat /K m values increased by 10%, 20%, 140%, and 100%, respectively, compared to those of the wild-type enzyme. Mechanisms that could account for these enhancements were explored. Moreover, in conjunction with the enzyme glucoamylase, the D503Y and D437H/D503Y mutants exhibited an improved reaction rate and glucose yield during starch hydrolysis compared to those of the wild-type enzyme, confirming the enhanced properties of the mutants. The mutants generated in this study have potential applications in the starch industry.

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Structure‐guided consensus approach to create a more thermostable penicillin G acylase

Cited by 76 publications

References 38 publications

Bridging the gaps in design methodologies by evolutionary optimization of the stability and proficiency of designed Kemp eliminase KE59

Bridging the gaps in design methodologies by evolutionary optimization of the stability and proficiency of designed Kemp eliminase KE59

Protein Engineering by Random Mutagenesis and Structure-Guided Consensus of Geobacillus stearothermophilus Lipase T6 for Enhanced Stability in Methanol

Improving the Thermostability and Catalytic Efficiency of Bacillus deramificans Pullulanase by Site-Directed Mutagenesis

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