In the light of directed evolution: Pathways of adaptive protein evolution

Bloom, Jesse D.; Arnold, Frances H.

doi:10.1073/pnas.0901522106

Cited by 400 publications

(375 citation statements)

References 66 publications

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“…It also was noteworthy that the GalT activity of the Vv GT5-R140W mutant was much lower than its GlcT activity and the fact that the T19P mutation of the Vv GT5-R140W mutant enhanced its GalT activity mimicked the evolutionary process of Vv GT6 for acquiring bifunctional specificity ( Figure 5). The bifunctionality of Vv GT6 can be regarded as a promiscuous state with respect to sugar donor specificity, as indicated by previous directed evolution studies (Aharoni et al, 2005;Bloom and Arnold, 2009), which show that enzymes tend to go through a promiscuous state prior to neofunctionalization.…”

Section: Evolution Of Sugar Donor Specificitymentioning

confidence: 95%

Functional Differentiation of the Glycosyltransferases That Contribute to the Chemical Diversity of Bioactive Flavonol Glycosides in Grapevines (Vitis vinifera)

et al. 2010

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We identified two glycosyltransferases that contribute to the structural diversification of flavonol glycosides in grapevine (Vitis vinifera): glycosyltransferase 5 (Vv GT5) and Vv GT6. Biochemical analyses showed that Vv GT5 is a UDP-glucuronic acid:flavonol-3-O-glucuronosyltransferase (GAT), and Vv GT6 is a bifunctional UDP-glucose/UDP-galactose:flavonol-3-Oglucosyltransferase/galactosyltransferase. The Vv GT5 and Vv GT6 genes have very high sequence similarity (91%) and are located in tandem on chromosome 11, suggesting that one of these genes arose from the other by gene duplication. Both of these enzymes were expressed in accordance with flavonol synthase gene expression and flavonoid distribution patterns in this plant, corroborating their significance in flavonol glycoside biosynthesis. The determinant of the specificity of Vv GT5 for UDP-glucuronic acid was found to be Arg-140, which corresponded to none of the determinants previously identified for other plant GATs in primary structures, providing another example of convergent evolution of plant GAT. We also analyzed the determinants of the sugar donor specificity of Vv GT6. Gln-373 and Pro-19 were found to play important roles in the bifunctional specificity of the enzyme. The results presented here suggest that the sugar donor specificities of these Vv GTs could be determined by a limited number of amino acid substitutions in the primary structures of protein duplicates, illustrating the plasticity of plant glycosyltransferases in acquiring new sugar donor specificities. INTRODUCTIONGrapevine (Vitis vinifera) ( Figure 1A) has been one of the most important fruit crops from the beginning of human civilization (McGovern et al., 1997). Dietary intake of grapevine-based products results in diverse biological effects that are beneficial to human health, in part due to polyphenols, such as flavonoids (e.g., flavonols, proanthocyanidins, and anthocyanins) (Renaud and de Lorgeril, 1992;Corder et al., 2001Corder et al., , 2006Baur et al., 2006). Flavonols, such as kaempferol, quercetin, and isorhamnetin, are an important class of bioactive flavonoids, which are most abundant as their 3-O-glycosides (i.e., glucoside, glucuronoside, galactoside, and rutinoside) in the berries, seeds, and leaves of grapevine (Hmamouchi et al., 1996;Castillo-Munoz et al., 2009).In this plant, biosynthesis of flavonol glycosides primarily serves to protect the plant from excess UV light (Price et al., 1995;Matus et al., 2009). Flavonol aglycons are biosynthesized from dihydroflavonols by the action of flavonol synthase (Vv FLS), a 2-oxoglutarate-dependent dioxygenase, which is temporally regulated by an R2R3-Myb transcription factor, Vv MYBF1 (Fujita et al., 2006;Czemmel et al., 2009). Subsequently, glycosylation of flavonols enhances their water solubility, such that high concentrations of flavonols can accumulate in plant cells.From a food chemistry perspective, flavonol glycosides define the color of wine grapes and products by acting as copigments (Yoshitama et al., 1992;Boulto...

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Section: Evolution Of Sugar Donor Specificitymentioning

confidence: 95%

Functional Differentiation of the Glycosyltransferases That Contribute to the Chemical Diversity of Bioactive Flavonol Glycosides in Grapevines (Vitis vinifera)

et al. 2010

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“…[55][56][57] In contrast, improving the efficiency at which an enzyme catalyzes a reaction with its native substrate is very difficult to achieve. 27,37,58 Despite the fact that paraoxon was only present in the environment for ca.…”

Section: Mechanistic Analysis Of Unnatural Phosphotriesterasesmentioning

confidence: 99%

Improving a Natural Enzyme Activity through Incorporation of Unnatural Amino Acids

Ugwumba

Ozawa

et al. 2010

J. Am. Chem. Soc.

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Abstract:The bacterial phosphotriesterases catalyze hydrolysis of the pesticide paraoxon with very fast turnover rates and are thought to be near to their evolutionary limit for this activity. To test whether the naturally evolved turnover rate could be improved through the incorporation of unnatural amino acids and to probe the role of peripheral active site residues in nonchemical steps of the catalytic cycle (substrate binding and product release), we replaced the naturally occurring tyrosine amino acid at position 309 with unnatural L- (7-hydroxycoumarin-4-yl)ethylglycine (Hco) and L-(7-methylcoumarin-4-yl)ethylglycine amino acids, as well as leucine, phenylalanine, and tryptophan. Kinetic analysis suggests that the 7-hydroxyl group of Hco, particularly in its deprotonated state, contributes to an increase in the rate-limiting product release step of substrate turnover as a result of its electrostatic repulsion of the negatively charged 4-nitrophenolate product of paraoxon hydrolysis. The 8-11-fold improvement of this already highly efficient catalyst through a single rationally designed mutation using an unnatural amino acid stands in contrast to the difficulty in improving this native activity through screening hundreds of thousands of mutants with natural amino acids. These results demonstrate that designer amino acids provide easy access to new and valuable sequence and functional space for the engineering and evolution of existing enzyme functions.

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“…Proteins are marginally stable and become nonfunctional if destabilized past a threshold. Thus, protein stability is linked to measures of protein function like catalytic activity or ligand binding (8)(9)(10). For example, a single mutation that decreases stability beyond the threshold can dramatically reduce protein function.…”

mentioning

confidence: 99%

A fundamental protein property, thermodynamic stability, revealed solely from large-scale measurements of protein function

Araya¹,

Fowler

Chen³

et al. 2012

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

240

264

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The ability of a protein to carry out a given function results from fundamental physicochemical properties that include the protein's structure, mechanism of action, and thermodynamic stability. Traditional approaches to study these properties have typically required the direct measurement of the property of interest, oftentimes a laborious undertaking. Although protein properties can be probed by mutagenesis, this approach has been limited by its low throughput. Recent technological developments have enabled the rapid quantification of a protein's function, such as binding to a ligand, for numerous variants of that protein. Here, we measure the ability of 47,000 variants of a WW domain to bind to a peptide ligand and use these functional measurements to identify stabilizing mutations without directly assaying stability. Our approach is rooted in the well-established concept that protein function is closely related to stability. Protein function is generally reduced by destabilizing mutations, but this decrease can be rescued by stabilizing mutations. Based on this observation, we introduce partner potentiation, a metric that uses this rescue ability to identify stabilizing mutations, and identify 15 candidate stabilizing mutations in the WW domain. We tested six candidates by thermal denaturation and found two highly stabilizing mutations, one more stabilizing than any previously known mutation. Thus, physicochemical properties such as stability are latent within these large-scale protein functional data and can be revealed by systematic analysis. This approach should allow other protein properties to be discovered.deep mutational scanning | epistasis | high-throughput DNA sequencing

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In the light of directed evolution: Pathways of adaptive protein evolution

Cited by 400 publications

References 66 publications

Functional Differentiation of the Glycosyltransferases That Contribute to the Chemical Diversity of Bioactive Flavonol Glycosides in Grapevines (Vitis vinifera)

Functional Differentiation of the Glycosyltransferases That Contribute to the Chemical Diversity of Bioactive Flavonol Glycosides in Grapevines (Vitis vinifera)

Improving a Natural Enzyme Activity through Incorporation of Unnatural Amino Acids

A fundamental protein property, thermodynamic stability, revealed solely from large-scale measurements of protein function

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