Laboratory-Directed Protein Evolution

Yuan, Ling; Kurek, Itzhak; English, James J.; Keenan, Robert J.

doi:10.1128/mmbr.69.3.373-392.2005

Cited by 177 publications

(98 citation statements)

References 365 publications

(329 reference statements)

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“…Directed evolution is an elegant method that is often used to alter enzymatic function (usually by broadening enzymatic specificity), but it also has been used to change the properties of one enzyme so that it has some of the functional properties of another related enzyme (2). One way to do this requires the presence of a unique and selectable phenotype for one family member, allowing the selection of gain-of-function mutations in a related protein.…”

mentioning

confidence: 99%

Laboratory evolution of one disulfide isomerase to resemble another

Hiniker

Ren

Heras

et al. 2007

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

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It is often difficult to determine which of the sequence and structural differences between divergent members of multigene families are functionally important. Here we use a laboratory evolution approach to determine functionally important structural differences between two distantly related disulfide isomerases, DsbC and DsbG from Escherichia coli. Surprisingly, we found single amino acid substitutions in DsbG that were able to complement dsbC in vivo and have more DsbC-like isomerase activity in vitro. Crystal structures of the three strongest point mutants, DsbG K113E, DsbG V216M, and DsbG T200M, reveal changes in highly surface-exposed regions that cause DsbG to more closely resemble the distantly related DsbC. In this case, laboratory evolution appears to have taken a direct route to allow one protein family member to complement another, with single substitutions apparently bypassing much of the need for multiple changes that took place over Ϸ0.5 billion years of evolution. Our findings suggest that, for these two proteins at least, regions important in determining functional differences may represent only a tiny fraction of the overall protein structure.chaperone ͉ protein folding ͉ directed evolution A number of models of the origin of life postulate that the primordial living cell contained only a small number of enzymes; years of gene duplication, divergence, and natural selection led to the current situation in which each organism possesses thousands of proteins with different functions (1). Identifying the mechanisms by which proteins can acquire new functions is important both in understanding this fundamental question of natural diversity and in elucidating the particular structural differences that allow related proteins to have distinct functions. Here we use a combination of directed evolution and structural biology to examine the functionally important structural differences of two distantly related Escherichia coli disulfide isomerases, DsbC and DsbG.Directed evolution is an elegant method that is often used to alter enzymatic function (usually by broadening enzymatic specificity), but it also has been used to change the properties of one enzyme so that it has some of the functional properties of another related enzyme (2). One way to do this requires the presence of a unique and selectable phenotype for one family member, allowing the selection of gain-of-function mutations in a related protein. Analysis of those gain-of-function mutants can provide information about functional differences between the two family members. This technique has been used to alter the enzymatic activity of a number of proteins so that they now have common properties, including the E. coli paralogs aspartate aminotransferase (AATase) and tyrosine aminotransferase (3), the structurally similar HisA and TrpF (4), and the human class 1-1 and rat class 2-2 glutathione transferases (5). Directed evolution has also been used to explore the differences between various E. coli folding catalysts, such as DsbC and DsbA (6), Gr...

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confidence: 99%

Laboratory evolution of one disulfide isomerase to resemble another

Hiniker

Ren

Heras

et al. 2007

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

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“…Directed evolution is a proven effective process for protein engineering and optimization (25,26). Within the short time scale, a remarkable range of new enzymatic properties can be generated, based on creating different libraries and adopting different screening approaches.…”

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Engineering Monolignol 4-O-Methyltransferases to Modulate Lignin Biosynthesis

Bhuiya

Liu

2010

Journal of Biological Chemistry

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Lignin is a complex polymer derived from the oxidative coupling of three classical monolignols. Lignin precursors are methylated exclusively at the meta-positions (i.e. 3/5-OH) of their phenyl rings by native O-methyltransferases, and are precluded from substitution of the para-hydroxyl (4-OH) position. Ostensibly, the para-hydroxyls of phenolics are critically important for oxidative coupling of phenoxy radicals to form polymers. Therefore, creating a 4-O-methyltransferase to substitute the para-hydroxyl of monolignols might well interfere with the synthesis of lignin. The phylogeny of plant phenolic O-methyltransferases points to the existence of a batch of evolutionarily "plastic" amino acid residues. Following one amino acid at a time path of directed evolution, and using the strategy of structure-based iterative site-saturation mutagenesis, we created a novel monolignol 4-O-methyltransferase from the enzyme responsible for methylating phenylpropenes. We show that two plastic residues in the active site of the parental enzyme are vital in dominating substrate discrimination. Mutations at either one of these separate the evolutionarily tightly linked properties of substrate specificity and regioselective methylation of native O-methyltransferase, thereby conferring the ability for para-methylation of the lignin monomeric precursors, primarily monolignols. Beneficial mutations at both sites have an additive effect. By further optimizing enzyme activity, we generated a triple mutant variant that may structurally constitute a novel phenolic substrate binding pocket, leading to its high binding affinity and catalytic efficiency on monolignols. The 4-O-methoxylation of monolignol efficiently impairs oxidative radical coupling in vitro, highlighting the potential for applying this novel enzyme in managing lignin polymerization in planta.

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“…These include oligonucleotidedirected randomization, whole gene randomization, homology-dependent recombination, and homologyindependent combination methods. I refer the reader to many excellent reviews and references therein for more comprehensive coverage (Farinas et al, 2001;Kurtzman et al, 2001;Lutz and Patrick, 2004;Neylon, 2004;Yuan et al, 2005). The success of DNA shuffling notwithstanding, perhaps the greatest disadvantage of the method is the high rate of parental background.…”

Section: Molecular Evolution Strategiesmentioning

confidence: 99%

The Outlook for Protein Engineering in Crop Improvement

Rao

2008

Plant Physiology

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Laboratory-Directed Protein Evolution

Cited by 177 publications

References 365 publications

Laboratory evolution of one disulfide isomerase to resemble another

Laboratory evolution of one disulfide isomerase to resemble another

Engineering Monolignol 4-O-Methyltransferases to Modulate Lignin Biosynthesis

The Outlook for Protein Engineering in Crop Improvement

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