Evolutionary Optimization of Computationally Designed Enzymes: Kemp Eliminases of the KE07 Series

Khersonsky, Olga; Röthlisberger, Daniela; Dym, Orly; Albeck, Shira; Jackson, Colin J.; Baker, David; Tawfik, Dan S.

doi:10.1016/j.jmb.2009.12.031

Cited by 161 publications

(200 citation statements)

References 42 publications

Supporting

Mentioning

187

Contrasting

Unclassified

Order By: Relevance

“…S7). In the case of KE07, directed evolution led to a significant increase in the basicity (≥1.7 pK a units) and reactivity of its catalytic Glu (16). For KE59, the increase in E230's basicity was smaller [around 0.6 units for pK a (k cat )].…”

Section: Resultsmentioning

confidence: 99%

“…The marginal stability of proteins restricts the accumulation of mutations, and especially of mutations that modulate protein function (19,20). KE07 and KE70 designs possessed an excess of stability that enabled mutations to accumulate (16). However, as is the case with many natural proteins, the stability of KE59 was limited, and so was its evolvability-no improved variants were observed in libraries of random mutations.…”

Section: Discussionmentioning

confidence: 99%

“…The KE07 design was optimized by directed evolution (16), and KE70 by a combination of directed evolution and computational design (17). The k cat ∕K m values of optimized KE07 and KE70 variants (2.4 × 10 3 -5 × 10 4 M −1 s −1 ) compare well with other enzyme mimics but are still inferior to natural enzymes.…”

mentioning

confidence: 99%

See 2 more Smart Citations

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.

Self Cite

214

221

View full text Add to dashboard Cite

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...

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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.

Self Cite

214

221

View full text Add to dashboard Cite

show abstract

“…Directed evolution has been shown to be an effective strategy to increase the activity of designed enzymes (2,21,26) and may offer insight into the deficiencies in the design.…”

Section: Discussionmentioning

confidence: 99%

Iterative approach to computational enzyme design

Privett

Kiss

Lee

et al. 2012

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

295

357

View full text Add to dashboard Cite

A general approach for the computational design of enzymes to catalyze arbitrary reactions is a goal at the forefront of the field of protein design. Recently, computationally designed enzymes have been produced for three chemical reactions through the synthesis and screening of a large number of variants. Here, we present an iterative approach that has led to the development of the most catalytically efficient computationally designed enzyme for the Kemp elimination to date. Previously established computational techniques were used to generate an initial design, HG-1, which was catalytically inactive. Analysis of HG-1 with molecular dynamics simulations (MD) and X-ray crystallography indicated that the inactivity might be due to bound waters and high flexibility of residues within the active site. This analysis guided changes to our design procedure, moved the design deeper into the interior of the protein, and resulted in an active Kemp eliminase, HG-2. The cocrystal structure of this enzyme with a transition state analog (TSA) revealed that the TSA was bound in the active site, interacted with the intended catalytic base in a catalytically relevant manner, but was flipped relative to the design model. MD analysis of HG-2 led to an additional point mutation, HG-3, that produced a further threefold improvement in activity. This iterative approach to computational enzyme design, including detailed MD and structural analysis of both active and inactive designs, promises a more complete understanding of the underlying principles of enzymatic catalysis and furthers progress toward reliably producing active enzymes.computational protein design | de novo enzyme design | proton transfer T he high efficiency, chemoselectivity, regio-and stereospecificity, and biodegradability of enzymes make them extremely attractive catalysts. However, the finite repertoire of naturally occurring enzymes limits their applicability to broad problems in biotechnology. A general method for the computational design of enzymes that can efficiently catalyze arbitrary chemical reactions would allow the benefits of enzymatic catalysis to be applied to chemical transformations of interest that are currently inaccessible via natural enzymes. Bolon and Mayo provided important early evidence that such an approach is feasible (1), which motivated significant progress toward this goal in recent years. Using quantum mechanics-based active site design and the Rosetta software suite, Baker, Houk, and coworkers designed enzymes for three chemically unrelated nonnatural reactions in a variety of catalytically inert scaffolds (2-4).In early incarnations of computational protein design, a strategy for methods development was put forth in terms of the so-called "protein design cycle" in which experimental evaluation of an initial design is used to inform adjustments to the design process for subsequent rounds of design (5, 6). Ideally, these steps would be continued iteratively until the protein sequences predicted by the algorithm exhibit the desired char...

show abstract

“…An aliquot of 135 µL of 0.1 M NaOH was added after 5 min and conversion to the respective product was determined by HPLC as decribed above. pH rate data were fitted for variants WT, Var8 and ILS6 using equation 1 [31] . VH = (Vmax•10^(-pKa,app))/(10^(-pH)+10^(-pKa,app))…”

Section: Expression and Purification Of Improved Idf And Ils Variantsmentioning

confidence: 99%

Directed Divergent Evolution of a Thermostable D‐Tagatose Epimerase towards Improved Activity for Two Hexose Substrates

et al. 2015

View full text Add to dashboard Cite

Abstract:We exploit the functional promiscuity of an engineered thermostable variant of a promiscuous D-tagatose epimerase (DTE Var8) to morph it into two efficient catalysts for the C3 epimerization of D-fructose to D-psicose and of L-sorbose to L-tagatose. Iterative single-site randomization and screening of 48 residues in the first and second shell around the substrate binding site of Var8 yielded 8-sites mutant IDF8 with an 9-fold improved kcat for the epimerization of D-fructose, and the 6-sites mutant ILS6 with an 14-fold improved epimerization of L-sorbose compared to Var8. Structure analysis of IDF8 revealed a charged patch at the entrance of its active site that supposedly facilitates the entry of the polar substrate, whereas the improvement in variant ILS6 is thought to relate to subtle changes in the hydration of the bound substrate. The structures will inform future engineering efforts of these and other isomerizing enzymes for the production of rare.

show abstract

Evolutionary Optimization of Computationally Designed Enzymes: Kemp Eliminases of the KE07 Series

Cited by 161 publications

References 42 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

Iterative approach to computational enzyme design

Directed Divergent Evolution of a Thermostable D‐Tagatose Epimerase towards Improved Activity for Two Hexose Substrates

Contact Info

Product

Resources

About