How Does an Enzyme Evolved In vitro Compare to Naturally Occurring Homologs Possessing the Targeted Function? Tyrosine Aminotransferase from Aspartate Aminotransferase

Rothman, Steven C.; Kirsch, Jack F.

doi:10.1016/s0022-2836(03)00095-0

Cited by 64 publications

(64 citation statements)

References 49 publications

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“…This allows for several generations of "generalist" proteins [11] that are able to perform both functions, and suggests that gene duplication acts after the new function has appeared [8] and not before.…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

Using multi-objective computational design to extend protein promiscuity

Suárez

Tortosa

Garcia-Mira

et al. 2010

Biophysical Chemistry

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Section: Accepted M Manuscriptmentioning

confidence: 99%

Using multi-objective computational design to extend protein promiscuity

Suárez

Tortosa

Garcia-Mira

et al. 2010

Biophysical Chemistry

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“…Enzymes evolved for higher substrate enantioselectivity often exhibit lower specific activities toward their new substrates relative to their respective parental enzymes (13)(14)(15). Similarly, the evolution of highly active variants of aspartate aminotransferases capable of accepting branched or aromatic amino acid substrates was accompanied by a relaxation of the substrate selectivity (16,17).In nature, the evolution of enzymes occurs as the result of positive selective pressure for turnover of physiological substrates, combined with simultaneous negative selective pressure to eliminate completely, or at least drastically suppress, deleterious activities. It follows that the engineering of enzymes exhibiting high catalytic activity and substrate selectivity for a particular desired substrate should be similarly accomplished by implementing selection and counterselection assay schemes in the laboratory.…”

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confidence: 99%

“…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.…”

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confidence: 99%

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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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“…[3][4][5][6] Mutagenesis studies have shown that the specificity of these enzymes can be modulated through the modification of relatively few residues. [7][8][9][10][11] For example, the HEX 7 and SRHEPT 8 mutants, which behave as TATases in terms of substrate specificity (w-TATase active sites), were obtained from rational redesign and directed evolution of eAATase, respectively. As their names indicate, they differ from eAATase by only six and seven point mutations per monomer.…”

Section: Introductionmentioning

confidence: 99%

Engineering homooligomeric proteins to detect weak intersite allosteric communication: Aminotransferases, a case study

Deu

Kirsch

2011

Protein Science

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The existence of low levels of intersubunit communication in homooligomeric enzymes is often difficult to discover, as the identical active sites cannot be probed individually to dissect their interdependent contributions. The homodimeric paralogs, E. coli aspartate-(AATase) and tyrosine aminotransferase (TATase), have not been demonstrated to show allostery. To address this question, we engineered a hybrid aminotransferase containing two distinct catalytic pockets: an AATase and a TATase site. The TATase/AATase hybrid was constructed by grafting an engineered TATase active site into one of the catalytic pockets of E. coli AATase. Each active site conserves its specific catalytic and inhibitor binding properties, and the hybrid catalyzes simultaneously each aminotransferase reaction at the respective site. Importantly, association of a selective inhibitor into one of the catalytic pockets decreases the activity of the second active site by up to 25%, thus proving unequivocally the existence of allosteric communication between active sites. The procedure may be applicable to other homologous sets of enzymes.

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How Does an Enzyme Evolved In vitro Compare to Naturally Occurring Homologs Possessing the Targeted Function? Tyrosine Aminotransferase from Aspartate Aminotransferase

Cited by 64 publications

References 49 publications

Using multi-objective computational design to extend protein promiscuity

Using multi-objective computational design to extend protein promiscuity

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

Engineering homooligomeric proteins to detect weak intersite allosteric communication: Aminotransferases, a case study

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