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Selection and evolution of enzymes from a partially randomized non-catalytic scaffold.

ABSTRACT: Enzymes are exceptional catalysts that facilitate a wide variety of reactions under mild conditions, achieving high rate-enhancements with excellent chemo-, regio- and stereoselectivities. There is considerable interest in developing new enzymes for the synthesis of chemicals and pharmaceuticals and as tools for molecular biology. Methods have been developed for modifying and improving existing enzymes through screening, selection and directed evolution. However, the design and evolution of truly novel enzymes has relied on extensive knowledge of the mechanism of the reaction. Here we show that genuinely new enzymatic activities can be created de novo without the need for prior mechanistic information by selection from a naive protein library of very high diversity, with product formation as the sole selection criterion. We used messenger RNA display, in which proteins are covalently linked to their encoding mRNA, to select for functional proteins from an in vitro translated protein library of >10(12 )independent sequences without the constraints imposed by any in vivo step. This technique has been used to evolve new peptides and proteins that can bind a specific ligand, from both random-sequence libraries and libraries based on a known protein fold. We now describe the isolation of novel RNA ligases from a library that is based on a zinc finger scaffold, followed by in vitro directed evolution to further optimize these enzymes. The resulting ligases exhibit multiple turnover with rate enhancements of more than two-million-fold.

SUBMITTER: Seelig B

PROVIDER: S-EPMC4476047 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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Selection and evolution of enzymes from a partially randomized non-catalytic scaffold.

Seelig Burckhard B Szostak Jack W JW

Nature 20070801 7155

Enzymes are exceptional catalysts that facilitate a wide variety of reactions under mild conditions, achieving high rate-enhancements with excellent chemo-, regio- and stereoselectivities. There is considerable interest in developing new enzymes for the synthesis of chemicals and pharmaceuticals and as tools for molecular biology. Methods have been developed for modifying and improving existing enzymes through screening, selection and directed evolution. However, the design and evolution of trul ...[more]

PMID: 17700701

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Project description:The (βa)8-barrel enzyme fold is frequently encountered in nature and catalyzes a multitude of different reactions. It is therefore interesting to evaluate the evolutionary potential of this fold by laboratory experiments. Along these lines, we tested the ability of the (βa)8-barrel to acquire new catalytic functions by an approach combining random mutagenesis and selection in vivo. The genes encoding 52 different phosphate-binding (βa)8-barrel proteins were randomized by error-prone PCR and cloned into an expression plasmid. The resulting mixed repertoire was used to transform different auxotrophic Escherichia coli (E. coli) strains, each lacking an enzyme with a phosphate-containing substrate. After plating of the different transformants on minimal medium, growth was observed only for two strains, lacking either the gene for the serine phosphatase SerB or the phosphoserine aminotransferase SerC. The same variants of point-mutated E. coli genes nanE (encoding a putative N-acetlymannosamine-6-phosphate 2-epimerase) and pdxJ (encoding the pyridoxine 5’-phosphate synthase) were responsible for rescuing both DserB and DserC. Remarkably, all selected PdxJ variants have acquired a 10-residue C-terminal extension from the cloning vector, that is essential for the rescue of DserB and DserC. Unexpectedly, the complementing NanE and PdxJ variants did not catalyze the SerB or Serc reactions in vitro. However, RT-qPCR, RNAseq, and transcriptome analysis showed that they rescue the deletions by enlisting the help of endogenous E. coli enzymes HisB and HisC through exclusive up-regulation of histidine operon transcription. While the promiscuous SerB activity of HisB is well-established, our data indicates that HisC is promiscuous for the SerC reaction, as well. The successful rescue of DserB and DserC through point mutations and recruitment of additional amino acids in NanE and PdxJ provides another example for the adaptability of the (βa)8-barrel fold. To our knowledge, this is the first example of (βa)8-barrels functioning not as enzymes, but by activating transcription via an unknown mechanism.

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Selection and evolution of enzymes from a partially randomized non-catalytic scaffold.

Publications

Selection and evolution of enzymes from a partially randomized non-catalytic scaffold.

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