Exploring the active site of chorismate mutase by combinatorial mutagenesis and selection: the importance of electrostatic catalysis.

Kast, Peter; Asif-Ullah, M.; Jiang, Ning; Hilvert, Donald

doi:10.1073/pnas.93.10.5043

Cited by 149 publications

(246 citation statements)

References 42 publications

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“…The simple metabolic selection (20) used in this work was not designed to differentiate mutants of various activities above a low threshold (8) (mutase activity 9,000-fold lower than that of mMjCM was sufficient to confer survival). It is possible, however, that protein NRR may enable proteins of improved activity to be evolved even though the vast majority of genes immediately after NRR diversification encode inactive proteins.…”

Section: Discussionmentioning

confidence: 99%

“…Standard PCR, restriction digestion, and DNA ligation methods were used to assemble selection plasmid pCM, which contains the following key components: (i) the p15A replication origin from pACYC184; (ii) tyrA and pheC genes as in pKIMP-UAUC (20); (iii) the ␤-lactamase gene from pBR322; (iv) the chloramphenicol acetyltransferase (CAT) gene from pACYC184 for expression as a C-terminal protein fusion (lacking its natural start codon) located immediately downstream of restriction sites for protein library cloning; and (v) a tac promoter upstream of the library cloning site. The library insertion site was created by using synthetic PCR primers containing AflIII and BstEII sites.…”

Section: Methodsmentioning

confidence: 99%

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Directed evolution of protein enzymes using nonhomologous random recombination

Bittker

Liu

et al. 2004

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

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We recently reported the development of nonhomologous random recombination (NRR) as a method for nucleic acid diversification and applied NRR to the evolution of DNA aptamers. Here, we describe a modified method, protein NRR, that enables proteins to access diversity previously difficult or impossible to generate. We investigated the structural plasticity of protein folds and the ability of helical motifs to function in different contexts by applying protein NRR and in vivo selection to the evolution of chorismate mutase (CM) enzymes. Functional CM mutants evolved using protein NRR contained many insertions, deletions, and rearrangements. The distribution of these changes was not random but clustered in certain regions of the protein. Topologically rearranged but functional enzymes also emerged from these studies, indicating that multiple connectivities can accommodate a functional CM active site and demonstrating the ability to generate new domain connectivities through protein NRR. Protein NRR was also used to randomly recombine CM and fumarase, an unrelated but also ␣-helical protein. Whereas the resulting library contained fumarase fragments in many contexts before functional selection, library members surviving selection for CM activity invariably contained a CM core with fumarase sequences found only at the termini or in one loop. These results imply that internal helical fragments cannot be swapped between these proteins without the loss of nearly all CM activity. Our findings suggest that protein NRR will be useful in probing the functional requirements of enzymes and in the creation of new protein topologies. D irected evolution has been used to alter (1-4) enzyme activity as well as to investigate sequence constraints on protein folding (5-7) and catalysis (8). The sequences and therefore the properties of proteins that emerge from directed evolution depend on the diversification method used to generate the protein library before screening or selection. Theoretical studies (9) suggest that diversification methods, including point mutagenesis, homologous recombination, secondary structure swapping, and nonhomologous recombination differ in their ability to access protein sequences representing new folds and improved functions. Of the methods listed above, nonhomologous recombination has been theorized to be the most effective at enabling new structures and functions to emerge during protein evolution. Additional studies on enzyme superfamilies suggest that the reorganization of structurally similar components can result in altered substrate specificity or function (10). Because these components often lack DNA sequence homology, their combinatorial diversification can in general only be accomplished through nonhomologous recombination.Methods that enable recombination to take place at defined sites without sequence homology have been recently described (11). For example, it is possible to recombine unrelated proteinencoding genes by using synthetic oligonucleotides to encode each desired crossover (12, 1...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Directed evolution of protein enzymes using nonhomologous random recombination

Bittker

Liu

et al. 2004

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

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“…coli strains E. coli strains KA12 (genotype: D(srlR-recA)306: Tn10, D(pheA-tyrA-aroF), thi-1, endA-1, hsdR17, D(argF-lac)U169, supE44), 29 and XL1-Blue (Stratagene) were used for cloning purposes. KA13, a derivative of KA12 carrying a T7 RNA polymerase 27 was used for protein production, and KA12/pKIMP-UAUC 29 for in vivo selection experiments.…”

Section: Methodsmentioning

confidence: 99%

“…However, mixing the components at equimolar concentrations (0.1 lM) produced no significant CM activity in vitro (data not shown). Furthermore, when the genes for MjCM * 22-93 and a representative variant (Glu3Gly) of MjCM were subcloned into the selection plasmid pMG211 28 (yielding pMG211B_split, see Materials and Methods), coexpression in the CM-deficient E. coli selection strain KA12/pKIMP-UAUC 29 did not allow for growth on selective agar plates lacking phenylalanine and tyrosine (Supporting information Fig. 1).…”

Section: Design Of a Heterodimeric Chorismate Mutasementioning

confidence: 99%

“…29 Briefly, KA12/pKIMP-UAUC cells harboring target plasmids (pMG211B_hdCM, pMG211B_split, pMG211_mMjCM, pMG211_clone42S, pMG211_hdN1, pMG211_hdC3 or the plasmid library) were plated on M9c-agar plates 47 containing 100 lg/mL amp, 20 lg/mL cam, 60 lM IPTG (promoting gene expression from the helper plasmid pKIMP-UAUC), and 10 lM salicylate (to induce CM production) in the presence or absence of 20 lg/mL L-tyrosine and L-phenylalanine. For in vivo selection of library clones, 5 mL LB amp/ cam were inoculated with 50 lL of the glycerol stock and incubated until an OD 600 of 1 was reached.…”

Section: In Vivo Complementation Experimentsmentioning

confidence: 99%

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Design, selection, and characterization of a split chorismate mutase

et al. 2010

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Split proteins are versatile tools for detecting protein-protein interactions and studying protein folding. Here, we report a new, particularly small split enzyme, engineered from a thermostable chorismate mutase (CM). Upon dissecting the helical-bundle CM from Methanococcus jannaschii into a short N-terminal helix and a 3-helix segment and attaching an antiparallel leucine zipper dimerization domain to the individual fragments, we obtained a weakly active heterodimeric mutase. Using combinatorial mutagenesis and in vivo selection, we optimized the short linker sequences connecting the leucine zipper to the enzyme domain. One of the selected CMs was characterized in detail. It spontaneously assembles from the separately inactive fragments and exhibits wild-type like CM activity. Owing to the availability of a well characterized selection system, the simple 4-helix bundle topology, and the small size of the N-terminal helix, the heterodimeric CM could be a valuable scaffold for enzyme engineering efforts and as a split sensor for specifically oriented protein-protein interactions.

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Genetische Selektion - eine Strategie zur Untersuchung und Herstellung von Enzymen

2001

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Natürliche Enzyme sind über Jahrmillionen durch den graduellen Prozess der darwinistischen Evolution entstanden. Die Grundelemente der Evolution – Mutation, Selektion und Vervielfältigung – können auch im Labor eingesetzt werden, um Proteinkatalysatoren in entsprechend kürzeren Zeiträumen herzustellen und zu charakterisieren. Genetische In‐vivo‐Selektionsstrategien ermöglichen eine erschöpfende Analyse von Proteinbibliotheken mit 1010 unterschiedlichen Mitgliedern; und mit In‐vitro‐Methoden lassen sich sogar noch größere Ensembles studieren. Folglich können evolutionäre Ansätze statistisch wertvolle Einsichten in komplexe und oftmals subtile Wechselwirkungen geben, die die Proteinfaltung, die Struktur und den katalytischen Mechanismus beeinflussen. Solche Methoden werden zunehmend auch als Hilfsmittel beim Proteindesign eingesetzt, da sie den Zugang zu neuen Proteinen mit maßgeschneiderten katalytischen Aktivitäten und Selektivitäten eröffnen.

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Exploring the active site of chorismate mutase by combinatorial mutagenesis and selection: the importance of electrostatic catalysis.

Cited by 149 publications

References 42 publications

Directed evolution of protein enzymes using nonhomologous random recombination

Directed evolution of protein enzymes using nonhomologous random recombination

Design, selection, and characterization of a split chorismate mutase

Genetische Selektion - eine Strategie zur Untersuchung und Herstellung von Enzymen

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