Improved biocatalysts by directed evolution and rational protein design

Bornscheuer, Uwe T.; Pohl, Martina

doi:10.1016/s1367-5931(00)00182-4

Cited by 414 publications

(237 citation statements)

References 57 publications

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“…To first focus on C-5 and C-6, the set included a single C-5 challenge [L-altrose (Table 1; 16, the C-5-epimer of D-galactose)] and three C-6 challenges [6-deoxy-D-galactose (22, D-fucose), 6-amino-6-deoxy-D-galactose (23), and D-galacturonic acid (24)]. Of this set, only D-fucose (22) showed any turnover with wild-type GalK, albeit 2.7% of the turnover rate observed for D-galactose (14) (21). The graphical display of a typical screening result is given in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The application of E p rational engineering led to active site mutants capable of accepting a variety of substrates not used by the wild-type enzyme (9-11). The alternative to rational engineering is enzyme evolution, a process primarily dependent on the availability of a selection or high-throughput screen for the desired enhanced or altered enzymatic properties (22)(23)(24). With respect to carbohydrate enzymology, recent applications include the tagatose-1,6-bisphosphate aldolase modified by in vitro evolution toward an unnatural stereoselectivity (25), an evolved N-acetylneuraminic acid aldolase for L-sialic acid synthesis (26), or a 2-deoxy-D-ribose-5-phosphate aldolase with an expanded substrate range after site-directed mutagenesis (27).…”

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confidence: 99%

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Creation of the first anomeric D/L-sugar kinase by means of directed evolution

Hoffmeister

Yang²,

Liu³

et al. 2003

Proceedings of the National Academy of Sciences

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Chemoenzymatic routes toward complex glycoconjugates often depend on the availability of sugar-1-phosphates. Yet the chemical synthesis of these vital components is often tedious, whereas natural enzymes capable of anomeric phosphorylation are known to be specific for one or only a few monosaccharides. Herein we describe the application of directed evolution and a high-throughput multisugar colorimetric screen to enhance the catalytic capabilities of the Escherichia coli galactokinase GalK. From this approach, one particular GalK mutant carrying a single amino acid exchange (Y371H) displayed a surprisingly substantial degree of kinase activity toward sugars as diverse as D-galacturonic acid, D-talose, L-altrose, and L-glucose, all of which failed as wild-type GalK substrates. Furthermore, this mutant provides enhanced turnover of the small pool of sugars converted by the wild-type enzyme. Comparison of this mutation to the recently solved structure of Lactococcus lactis GalK begins to provide a blueprint for further engineering of this vital class of enzyme. In addition, the rapid access to such promiscuous sugar C-1 kinases will significantly enhance accessibility to natural and unnatural sugar-1-phosphates and thereby impact both in vitro and in vivo glycosylation methodologies, such as natural product glycorandomization.galactokinase ͉ glycorandomization ͉ in vitro evolution ͉ enzyme M any clinically important medicines are derived from glycosylated natural products, the D-or L-sugar substituents of which often dictate their overall biological activity. This paradigm is found throughout the anticancer and antiinfective arenas with representative clinical examples (Fig. 1a), including enediynes (calicheamicin, 1), polyketides (doxorubicin, 2; erythromycin, 3), indolocarbazoles (staurosporine, 4), nonribosomal peptides (vancomycin, 5), polyenes (nystatin, 6), coumarins (novobiocin, 7), and cardiac glycosides (digitoxin, 8) (1-7). Given the importance of the sugars attached to these and other biologically significant metabolites, extensive effort has been directed in recent years toward altering sugars as a means to enhance or alter natural product-based therapeutics by both in vivo and in vitro approaches (ref. 8 and references therein). Among these, in vitro glycorandomization (IVG) makes use of the inherent or engineered substrate promiscuity of nucleotidylyltransferases and glycosyltransferases to activate and attach chemically synthesized sugar precursors to various natural product scaffolds (6,7,(9)(10)(11)(12)(13)(14)(15), the advantage of which is the ability to efficiently incorporate highly functionalized ''unnatural'' sugar substitutions into the corresponding natural product scaffold (Fig. 1b). In a recent demonstration of IVG, Ͼ50 analogs of 5 were generated, some of which displayed enhanced and distinct antibacterial profiles from the parent natural product (13).As starting materials, sugar phosphates play a key role in the entire IVG process. Thus, the ability to rapidly construct sugar phosphate ...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Creation of the first anomeric D/L-sugar kinase by means of directed evolution

Hoffmeister

Yang²,

Liu³

et al. 2003

Proceedings of the National Academy of Sciences

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“…This method has proven to be efficient for the improvement of a variety of enzymatic properties (Bornscheuer and Pohl, 2001;Schmidt et al, 2009;Voigt et al, 2001).…”

Section: Discussionmentioning

confidence: 99%

Mutations at the putative active cavity of styrene monooxygenase: Enhanced activity and reversed enantioselectivity

Lin

Tang

Ahmed

et al. 2012

Journal of Biotechnology

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“…Two different but complementary technologies have been applied to this goal: (i) rational design, which relies on the availability of the three-dimensional structure and knowledge about the relationship between sequence, structure, and mechanism, and (ii) directed evolution methods, which use random mutagenesis of the gene encoding the protein or recombination of gene fragments to create diversity and then experimental screening of the libraries generated for the desired properties. Rational design has been used to elucidate and change enzyme mechanism, substrate and product specificity, enantioselectivity, cofactor specificity, and protein stability (1)(2)(3). Directed evolution has been applied to increase catalytic activity; to invert or improve enantioselectivity; and to alter substrate and product specificity, protein stability, pH optimum, and tolerance against organic solvents (1, 4 -7).…”

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confidence: 99%

“…In this study, we engineered the flavoenzyme vanillyl-alcohol oxidase (VAO) 1 by random mutagenesis such that the evolved mutants are capable of producing natural vanillin (4-hydroxy-3-methoxybenzaldehyde) from the precursor creosol (2-methoxy-4-methylphenol). Vanillin is a widely used flavor compound in food and personal products, with an estimated annual worldwide consumption of over 2000 tons (8,9).…”

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confidence: 99%

Laboratory-evolved Vanillyl-alcohol Oxidase Produces Natural Vanillin

Heuvel

Berg

Rovida

et al. 2004

Journal of Biological Chemistry

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The flavoenzyme vanillyl-alcohol oxidase was subjected to random mutagenesis to generate mutants with enhanced reactivity to creosol (2-methoxy-4-methylphenol). The vanillyl-alcohol oxidase-mediated conversion of creosol proceeds via a two-step process in which the initially formed vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) is oxidized to the widely used flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde). The first step of this reaction is extremely slow due to the formation of a covalent FAD N-5-creosol adduct. After a single round of error-prone PCR, seven mutants were generated with increased reactivity to creosol. The single-point mutants I238T, F454Y, E502G, and T505S showed an up to 40-fold increase in catalytic efficiency (k cat /K m ) with creosol compared with the wild-type enzyme. This enhanced reactivity was due to a lower stability of the covalent flavin-substrate adduct, thereby promoting vanillin formation. The catalytic efficiencies of the mutants were also enhanced for other ortho-substituted 4-methylphenols, but not for p-cresol (4-methylphenol). The replaced amino acid residues are not located within a distance of direct interaction with the substrate, and the determined three-dimensional structures of the mutant enzymes are highly similar to that of the wild-type enzyme. These results clearly show the importance of remote residues, not readily predicted by rational design, for the substrate specificity of enzymes.The increased use of enzymes and other proteins in the pharmaceutical, chemical, and agricultural industry has generated considerable interest in the design of proteins with new or improved properties. Two different but complementary technologies have been applied to this goal: (i) rational design, which relies on the availability of the three-dimensional structure and knowledge about the relationship between sequence, structure, and mechanism, and (ii) directed evolution methods, which use random mutagenesis of the gene encoding the protein or recombination of gene fragments to create diversity and then experimental screening of the libraries generated for the desired properties. Rational design has been used to elucidate and change enzyme mechanism, substrate and product specificity, enantioselectivity, cofactor specificity, and protein stability (1-3). Directed evolution has been applied to increase catalytic activity; to invert or improve enantioselectivity; and to alter substrate and product specificity, protein stability, pH optimum, and tolerance against organic solvents (1, 4 -7). An obvious advantage of directed evolution methods over sitedirected mutagenesis is that enzymes can be tailored for the production of (intermediate) products without detailed knowledge of protein structure and structure-function relationships.In this study, we engineered the flavoenzyme vanillyl-alcohol oxidase (VAO) 1 by random mutagenesis such that the evolved mutants are capable of producing natural vanillin (4-hydroxy-3-methoxybenzaldehyde) from the precursor creosol (2-methoxy...

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Improved biocatalysts by directed evolution and rational protein design

Cited by 414 publications

References 57 publications

Creation of the first anomeric D/L-sugar kinase by means of directed evolution

Creation of the first anomeric D/L-sugar kinase by means of directed evolution

Mutations at the putative active cavity of styrene monooxygenase: Enhanced activity and reversed enantioselectivity

Laboratory-evolved Vanillyl-alcohol Oxidase Produces Natural Vanillin

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