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2rkx
From Proteopedia
| 2rkx, resolution 2.25Å () | |
|---|---|
| Domains: | HisF, TIM_phosphate_binding |
| Related: | 1thf |
| Resources: | FirstGlance, OCA, PDBsum, RCSB |
| Coordinates: | save as pdb, mmCIF, xml |
Contents |
The 3D structure of a designed enzyme that acts as a Kemp elimination catalyst
The design of new enzymes for reactions not catalysed by naturally occurring biocatalysts is a challenge for protein engineering and is a critical test of our understanding of enzyme catalysis. Here we describe the computational design of eight enzymes that use two different catalytic motifs to catalyse the Kemp elimination-a model reaction for proton transfer from carbon-with measured rate enhancements of up to 105 and multiple turnovers. Mutational analysis confirms that catalysis depends on the computationally designed active sites, and a high-resolution crystal structure suggests that the designs have close to atomic accuracy. Application of in vitro evolution to enhance the computational designs produced a >200-fold increase in kcat/Km (kcat/Km of 2,600 M-1s-1 and kcat/kuncat of >106). These results demonstrate the power of combining computational protein design with directed evolution for creating new enzymes, and we anticipate the creation of a wide range of useful new catalysts in the future.
Kemp elimination catalysts by computational enzyme design., Rothlisberger D, Khersonsky O, Wollacott AM, Jiang L, DeChancie J, Betker J, Gallaher JL, Althoff EA, Zanghellini A, Dym O, Albeck S, Houk KN, Tawfik DS, Baker D, Nature. 2008 May 8;453(7192):190-5. Epub 2008 Mar 19. PMID:18354394
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
Comparison of the designed and crystal structures
We can take a look at a comparison of the and structures. The crystal structure (green) was solved in the unbound state and shows only modest rearrangement of side chains compared to the designed structure (red) modelled in the presence of the transition state (yellow). r.m.s.d. for the active site is 0.32 Å versus 0.95 Å for the active site including the .
Directed Evolution
of the wildtype (green) and catalytically improved directed evolutionary mutant (magenta) structures. In vitro evolution has been shown to markedly improve the stability, expression and activity of enzymes, and is currently the most widely used and successful approach for refining biocatalysts. The hydrophobic residue at the bottom of the active site was frequently mutated to polar or charged residues (the most common mutation being ), which may hold in position to stabilize the developing negative charge in the transition state while preventing interaction of Lys222 with . Consistent with this idea, the pKa of the catalytic Glu101 shifts from <4.5 to 5.9 in the evolved variant with the mutation.
About this Structure
2RKX is a Single protein structure. Full crystallographic information is available from OCA.
Primary Reference
Kemp elimination catalysts by computational enzyme design., Rothlisberger D, Khersonsky O, Wollacott AM, Jiang L, DeChancie J, Betker J, Gallaher JL, Althoff EA, Zanghellini A, Dym O, Albeck S, Houk KN, Tawfik DS, Baker D, Nature. 2008 May 8;453(7192):190-5. Epub 2008 Mar 19. PMID:18354394 Page seeded by OCA on Thu May 22 22:29:59 2008
