Design by Directed Evolution

Arnold, Frances H.

doi:10.1021/ar960017f

Cited by 622 publications

(428 citation statements)

References 24 publications

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“…Directed evolution is one of the most powerful tools presently available to improve the characteristics of enzymes (35)(36)(37). The success of the directed evolution approach depends on the efficient formation of a random mutant library and a highly quantitative and high-throughput screening system.…”

Section: Discussionmentioning

confidence: 99%

Directed Evolution of a Glycosynthase from Agrobacterium sp. Increases Its Catalytic Activity Dramatically and Expands Its Substrate Repertoire

Kim

Lee

Warren

et al. 2004

Journal of Biological Chemistry

122

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The Agrobacterium sp. ␤-glucosidase (Abg) is a retaining ␤-glycosidase and its nucleophile mutants, termed Abg glycosynthases, catalyze the formation of glycosidic bonds using ␣-glycosyl fluorides as donor sugars and various aryl glycosides as acceptor sugars. Two rounds of random mutagenesis were performed on the best glycosynthase to date (AbgE358G), and transformants were screened using an on-plate endocellulase coupled assay. Two highly active mutants were obtained, 1D12 (A19T, E358G) and 2F6 (A19T, E358G, Q248R, M407V) in the first and second rounds, respectively. Relative catalytic efficiencies (k cat /K m ) of 1:7:27 were determined for AbgE358G, 1D12, and 2F6, respectively, using ␣-D-galactopyranosyl fluoride and 4-nitrophenyl ␤-D-glucopyranoside as substrates. The 2F6 mutant is not only more efficient but also has an expanded repertoire of acceptable substrates. Analysis of a homology model structure of 2F6 indicated that the A19T and M407V mutations do not interact directly with substrates but exert their effects by changing the conformation of the active site. Much of the improvement associated with the A19T mutation seems to be caused by favorable interactions with the equatorial C2-hydroxyl group of the substrate. The alteration of torsional angles of Glu-411, Trp-412, and Trp-404, which are components of the aglycone (؉1) subsite, is an expected consequence of the A19T and M407V mutations based on the homology model structure of 2F6.Oligosaccharides have considerable potential as therapeutics because of the numerous medicinally relevant physiological events that involve glycoconjugates (1-3). To expand our understanding of the various roles of oligosaccharides found in important cellular events, more efficient and selective synthetic protocols must be developed for the preparation of oligosaccharides. Classical chemical synthesis is often impractical for the synthesis of complex oligosaccharides because of the need for selective and labor-intensive protection-deprotection steps and difficulties in directing product stereochemistry. To overcome these limitations, enzymatic syntheses using glycosidases or glycosyl transferases have rapidly gained prominence (4 -6).In recent years, the glycosynthase approach developed in this laboratory has added a new dimension to the enzymatic preparation of oligosaccharides (7-9). Glycosynthases are retaining glycosidase mutants in which the catalytic nucleophile has been converted to a non-nucleophilic residue. These mutants catalyze the formation of glycosidic bonds when glycosyl fluorides with anomeric configuration opposite to that of the original substrate, thereby mimicking the glycosyl enzyme intermediate, are employed as substrates. The modified enzyme catalyzes the nucleophilic displacement of the fluoride via attack by a hydroxyl group on an added glycosyl acceptor, generating a new glycosidic bond with the same stereochemistry as the normal substrate. The reactions catalyzed by glycosynthases are highly amenable to industrial syntheses because of the hig...

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Section: Discussionmentioning

confidence: 99%

Directed Evolution of a Glycosynthase from Agrobacterium sp. Increases Its Catalytic Activity Dramatically and Expands Its Substrate Repertoire

Kim

Lee

Warren

et al. 2004

Journal of Biological Chemistry

122

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“…6,7 Scientists began to use evolutionary strategies (Directed Evolution) 8 to tailor the properties of individual molecules. Random mutations or recombination can, in many cases, be done efficiently, leading in this way to molecular evolution in the laboratory.…”

Section: Introductionmentioning

confidence: 99%

Enzyme Promiscuity in Enolase Superfamily. Theoretical Study of o-Succinylbenzoate Synthase Using QM/MM Methods

et al. 2015

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The promiscuous activity of the enzyme o-Succinylbenzoate Synthase (OSBS) from the actinobacteria Amycolatopsis is investigated by means of QM/MM methods, using both Density Functional Theory and Semiempirical Hamiltonians. This enzyme catalyzes not only the dehydration of 2-succinyl-6R-hydroxy-2,4-cyclohexadiene-1R-carboxylate but also catalyzes racemization of different acylaminoacids, being N-succinyl-Rphenylglycine the best substrate. We investigated the molecular mechanisms for both reactions exploring the Potential Energy Surface. Then, Molecular Dynamics simulations were performed to obtain the free energy profiles and the averaged interaction energies of enzymatic residues with the reacting system. Our results confirm the plausibility of the reaction mechanisms proposed in the literature, with a good agreement between theoretical and experimentally-derived activation free energies. Our simulations unravel the role played by the different residues in each of the two possible reactions. The presence of flexible loops in the active site and the selection of structural modifications in the substrate seem to be key elements to promote the promiscuity of this enzyme.

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“…Mechanisms of protein stability in aqueous and nonaqueous environments 1,2 , the links between conformational mobility, structural integrity and activity 3,4 , and the complexities of substrate specificity have all succumbed to the onslaught of advanced molecular methods, including crystallography 5-7 , site-specific mutagenesis, gene shuffling, and protein evolution [8][9][10][11] . Scientists are now in a position to visualize, if not design, catalytic systems that approach the functional "ideal".…”

Section: Introductionmentioning

confidence: 99%

The search for the ideal biocatalyst

2002

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While the use of enzymes as biocatalysts to assist in the industrial manufacture of fine chemicals and pharmaceuticals has enormous potential, application is frequently limited by evolution-led catalyst traits. The advent of designer biocatalysts, produced by informed selection and mutation through recombinant DNA technology, enables production of process-compatible enzymes. However, to fully realize the potential of designer enzymes in industrial applications, it will be necessary to tailor catalyst properties so that they are optimal not only for a given reaction but also in the context of the industrial process in which the enzyme is applied.The past two decades have led to major advances in our understanding of the subtleties of protein structure-function interrelationships. Mechanisms of protein stability in aqueous and nonaqueous environments 1,2 , the links between conformational mobility, structural integrity and activity 3,4 , and the complexities of substrate specificity have all succumbed to the onslaught of advanced molecular methods, including crystallography 5-7 , site-specific mutagenesis, gene shuffling, and protein evolution [8][9][10][11] . Scientists are now in a position to visualize, if not design, catalytic systems that approach the functional "ideal".The "ideal" catalyst is typically considered by the biochemist in terms of turnover number (k cat ) or, for a given process, in terms of maximum specificity constant (A cat /KM). However, from a bioprocess viewpoint, each bioprocess is constrained by a set of conditions dictated by the specific properties of the substrates, products, and the bioconversion reaction. Thus, while for any given bioprocess it is clearly possible to specify a set of catalyst properties that would constitute the ideal for that process, only broad generalizations for ideality may otherwise be identified.In this review, we discuss molecular properties of enzymes from a bioprocess viewpoint. A paradigm for design based on the ideal process determining the desired features of the catalyst is presented. "Ideal" characteristics (generic and process-specific) and methodologies for seeking or engineering these are discussed. We then review the extent to which significant changes in protein functional properties have been achieved by application of these methods, together with issues that require more research and/or development. Ideal processesBioprocess engineers develop processes not only against fixed capital and operating expenditure, but also rapid, and often, very aggressive timelines. This is particularly the case for pharmaceutical products where patent expiry on the product demands a robust and scalable process with short development times 12 . Conventionally, once a synthetic route is fixed and the biocatalytic step defined, bioprocess design and operation are centered on the properties of the

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Design by Directed Evolution

Cited by 622 publications

References 24 publications

Directed Evolution of a Glycosynthase from Agrobacterium sp. Increases Its Catalytic Activity Dramatically and Expands Its Substrate Repertoire

Directed Evolution of a Glycosynthase from Agrobacterium sp. Increases Its Catalytic Activity Dramatically and Expands Its Substrate Repertoire

Enzyme Promiscuity in Enolase Superfamily. Theoretical Study of o-Succinylbenzoate Synthase Using QM/MM Methods

The search for the ideal biocatalyst

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