Rapid Evolution of β-Glucuronidase Specificity by Saturation Mutagenesis of an Active Site Loop

Geddie, Melissa L.; Matsumura, Ichiro

doi:10.1074/jbc.m401447200

Cited by 65 publications

(57 citation statements)

References 49 publications

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“…Numerous structure-guided and directed evolution strategies have been used in search of enzyme variants that exhibit high catalytic rates with poor or inactive substrates of the parental enzyme (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). As impressive as these successes have been, the engineering of enzymes that exhibit turnover rates and selectivities with new substrates comparable to their natural counterparts has proven quite a challenge, especially when considering those enzymes for which a genetic selection strategy is not possible.…”

mentioning

confidence: 99%

Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity

Varadarajan

Gam

Olsen

et al. 2005

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

137

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The exquisite selectivity and catalytic activity of enzymes have been shaped by the effects of positive and negative selection pressure during the course of evolution. In contrast, enzyme variants engineered by using in vitro screening techniques to accept novel substrates typically display a higher degree of catalytic promiscuity and lower total turnover in comparison with their natural counterparts. Using bacterial display and multiparameter flow cytometry, we have developed a novel methodology for emulating positive and negative selective pressure in vitro for the isolation of enzyme variants with reactivity for desired novel substrates, while simultaneously excluding those with reactivity toward undesired substrates. Screening of a large library of random mutants of the Escherichia coli endopeptidase OmpT led to the isolation of an enzyme variant, 1.3.19, that cleaved an Ala-Arg peptide bond instead of the Arg-Arg bond preferred by the WT enzyme. Variant 1.3.19 exhibited greater than three million-fold selectivity (-Ala-Arg-͞-Arg-Arg-) and a catalytic efficiency for AlaArg cleavage that is the same as that displayed by the parent for the preferred substrate, Arg-Arg. A single amino acid Ser223Arg substitution was shown to recapitulate completely the unique catalytic properties of the 1.3.19 variant. These results can be explained by proposing that this mutation acts to ''swap'' the P 1 Arg side chain normally found in WT substrate peptides with the 223Arg side chain in the S 1 subsite of OmpT.engineering ͉ flow cytometry T he reprogramming of enzyme catalytic activity and selectivity is a central issue in protein biochemistry and biotechnology. Numerous structure-guided and directed evolution strategies have been used in search of enzyme variants that exhibit high catalytic rates with poor or inactive substrates of the parental enzyme (1-19). As impressive as these successes have been, the engineering of enzymes that exhibit turnover rates and selectivities with new substrates comparable to their natural counterparts has proven quite a challenge, especially when considering those enzymes for which a genetic selection strategy is not possible.In particular, enzymes engineered through laboratory evolution involving in vitro catalytic assays have often been found lacking, either with respect to turnover rates or selectivity, relative to catalyst-substrate pairs isolated from natural sources. As a typical example, an extensive directed evolution program led to the isolation of Escherichia coli ␤-glucuronidase variants with significant ␤-galactosidase (10) or xylanosidase (11) activities, but nonetheless even the best clones exhibited k cat ͞K m values Ͼ1,000 times lower than those of naturally occurring enzymes such as the E. coli ␤-galactosidase or the Thermoanaerobacterium saccharolyticum ␤-xylosidase.This trend appears to be general. In a recent comprehensive study, Aaron et al. (12) demonstrated that the evolution of higher activity toward poor substrates did not impair the parental catalytic activity, and,...

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mentioning

confidence: 99%

Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity

Varadarajan

Gam

Olsen

et al. 2005

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

137

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“…11 The endpoint of the present study was to impart greater improvement in fewer rounds of directed evolution through the semi-rational site-saturation mutagenesis approach. [25][26][27] BGAL is a good model for directed evolution studies because it is amenable to high-throughput screening, and because its structure and function are well understood. The structure of pNPgal (native substrate) differs from that of pNP-fuc (novel substrate) by an oxygen atom on the C6 substituent (hydroxymethyl versus methyl).…”

Section: Resultsmentioning

confidence: 99%

“…17-24 Some employ site-saturation mutagenesis, in which a small number of active site residues are "randomized" (mutated randomly). [25][26][27][28][29] Experimental evolutionists generally base their methodological decisions upon intuition rather than any systematic understanding of adaptive protein evolution. Persuasive case studies all too often convince workers to employ techniques that fail to solve seemingly analogous problems.…”

Section: Introductionmentioning

confidence: 99%

Site-saturation Mutagenesis is more Efficient than DNA Shuffling for the Directed Evolution of β-Fucosidase from β-Galactosidase

Parikh

Matsumura

2005

Journal of Molecular Biology

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Protein engineers use a variety of mutagenic strategies to adapt enzymes to novel substrates. Directed evolution techniques (random mutagenesis and high-throughput screening) offer a systematic approach to the management of protein complexity. This sub-discipline was galvanized by the invention of DNA shuffling, a procedure that randomly recombines point mutations in vitro. In one influential study, Escherichia coli β-galactosidase (BGAL) variants with enhanced β-fucosidase activity (tenfold increase in k cat /K M in reactions with the novel para-nitrophenyl-β-D-fucopyranoside substrate; 39-fold decrease in reactivity with the "native" para-nitrophenyl-β-D-galactopyranoside substrate) were evolved in seven rounds of DNA shuffling and screening. Here, we show that a single round of site-saturation mutagenesis and screening enabled the identification of β-fucosidases that are significantly more active (180-fold increase in k cat /K M in reactions with the novel substrate) and specific (700,000-fold inversion of specificity) than the best variants in the previous study. Sitesaturation mutagenesis thus proved faster, less resource-intensive and more effective than DNA shuffling for this particular evolutionary pathway.

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“…When this hot spot was subjected to saturation mutagenesis, variants with further improvement or novel specificity were identified (228). Protein engineering using site-directed and/or saturation mutagenesis, guided by information generated from directed evolution, can be an extremely powerful approach to create novel functionalities (73,88,208,316).…”

Section: Directed Evolution Of Pharmaceuticalsmentioning

confidence: 99%

Laboratory-Directed Protein Evolution

Yuan

Kurek

English

et al. 2005

Microbiol Mol Biol Rev

175

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Systematic approaches to directed evolution of proteins have been documented since the 1970s. The ability to recruit new protein functions arises from the considerable substrate ambiguity of many proteins. The substrate ambiguity of a protein can be interpreted as the evolutionary potential that allows a protein to acquire new specificities through mutation or to regain function via mutations that differ from the original protein sequence. All organisms have evolutionarily exploited this substrate ambiguity. When exploited in a laboratory under controlled mutagenesis and selection, it enables a protein to “evolve” in desired directions. One of the most effective strategies in directed protein evolution is to gradually accumulate mutations, either sequentially or by recombination, while applying selective pressure. This is typically achieved by the generation of libraries of mutants followed by efficient screening of these libraries for targeted functions and subsequent repetition of the process using improved mutants from the previous screening. Here we review some of the successful strategies in creating protein diversity and the more recent progress in directed protein evolution in a wide range of scientific disciplines and its impacts in chemical, pharmaceutical, and agricultural sciences

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Rapid Evolution of β-Glucuronidase Specificity by Saturation Mutagenesis of an Active Site Loop

Cited by 65 publications

References 49 publications

Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity

Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity

Site-saturation Mutagenesis is more Efficient than DNA Shuffling for the Directed Evolution of β-Fucosidase from β-Galactosidase

Laboratory-Directed Protein Evolution

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