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Directed evolution of protein enzymes using nonhomologous random recombination.

ABSTRACT: 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 alpha-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.

SUBMITTER: Bittker JA

PROVIDER: S-EPMC406457 | biostudies-literature | 2004 May

REPOSITORIES: biostudies-literature

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Directed evolution of protein enzymes using nonhomologous random recombination.

Bittker Joshua A JA Le Brian V BV Liu Jane M JM Liu David R DR

Proceedings of the National Academy of Sciences of the United States of America 20040426 18

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 evol ...[more]

PMID: 15118093

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Project description:BACKGROUND:Saccharification of lignocellulosic material by xylanases and other glycoside hydrolases is generally conducted at high concentrations of the final reaction products, which frequently inhibit the enzymes used in the saccharification process. Using a random nonhomologous recombination strategy, we have fused the GH11 xylanase from Bacillus subtilis (XynA) with the xylose binding protein from Escherichia coli (XBP) to produce an enzyme that is allosterically stimulated by xylose. RESULTS:The pT7T3GFP_XBP plasmid containing the XBP coding sequence was randomly linearized with DNase I, and ligated with the XynA coding sequence to create a random XynA-XBP insertion library, which was used to transform E. coli strain JW3538-1 lacking the XBP gene. Screening for active XBP was based on the expression of GFP from the pT7T3GFP_XBP plasmid under the control of a xylose inducible promoter. In the presence of xylose, cells harboring a functional XBP domain in the fusion protein (XBP+) showed increased GFP fluorescence and were selected using FACS. The XBP+ cells were further screened for xylanase activity by halo formation around xylanase producing colonies (XynA+) on LB-agar-xylan media after staining with Congo red. The xylanase activity ratio with xylose/without xylose in supernatants from the XBP+/XynA+ clones was measured against remazol brilliant blue xylan. A clone showing an activity ratio higher than 1.3 was selected where the XynA was inserted after the asparagine 271 in the XBP, and this chimera was denominated as XynA-XBP271. The XynA-XBP271 was more stable than XynA at 55 °C, and in the presence of xylose the catalytic efficiency was ~3-fold greater than the parental xylanase. Molecular dynamics simulations predicted the formation of an extended protein-protein interface with coupled movements between the XynA and XBP domains. In the XynA-XBP271 with xylose bound to the XBP domain, the mobility of a ?-loop in the XynA domain results in an increased access to the active site, and may explain the observed allosteric activation. CONCLUSIONS:The approach presented here provides an important advance for the engineering enzymes that are stimulated by the final product.

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Directed evolution of protein enzymes using nonhomologous random recombination.

Publications

Directed evolution of protein enzymes using nonhomologous random recombination.

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