Novel Enzymes Through Design and Evolution

Woycechowsky, Kenneth J.; Vamvaca, Katherina; Hilvert, Donald

doi:10.1002/9780471224464.ch4

Cited by 9 publications

(8 citation statements)

References 148 publications

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“…Rather it is performed by creating variants using error-prone polymerase chain reaction, saturation mutagenesis, or recombination followed by screening and selection variants for desired characteristics, such as enhanced activity, stability, or expression, or for altered substrate specificity [87-89]. Directed evolution has been successfully applied to the design of industrial biocatalysts for enhanced catalytic efficiency, novel activity, and increased stability [94-95]. The main requirement of directed evolution is to develop a high throughput activity screening (HTS) system to screen/select mutants with desired properties (Table 2).…”

Section: Approaches To Engineer Cytochrome P450mentioning

confidence: 99%

Engineering cytochrome P450 biocatalysts for biotechnology, medicine and bioremediation

Kumar

2010

Expert Opinion on Drug Metabolism & Toxicology

147

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Importance of the field: Cytochrome P450 enzymes comprise a superfamily of heme monooxygenases that are of considerable interest for the: 1) synthesis of novel drugs and drug metabolites, 2) targeted cancer gene therapy, 3) biosensor design, and 4) bioremediation. However, their applications are limited because cytochrome P450, especially mammalian P450 enzymes, show a low turnover rate and stability, and require a complex source of electrons through cytochrome P450 reductase and NADPH. Areas covered in this review: In this review, we discuss the recent progress towards the use of P450 enzymes in a variety of above-mentioned applications. We also present alternate and cost-effective ways to perform P450-mediated reaction, especially using peroxides. Furthermore, we expand upon the current progress in P450 engineering approaches describing several recent examples that are utilized to enhance heterologous expression, stability, catalytic efficiency, and utilization of alternate oxidants. What the reader will gain: The review will provide a comprehensive knowledge in the design of P450 biocatalysts for potentially practical purposes. Finally, we provide a prospective on the future aspects of P450 engineering and its applications in biotechnology, medicine, and bioremediation. Take home message: Because of its wide applications, academic and pharmaceutical researchers, environmental scientists, and health care providers are expected to gain current knowledge and future prospects of the practical use of P450 biocatalysts.

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Section: Approaches To Engineer Cytochrome P450mentioning

confidence: 99%

Engineering cytochrome P450 biocatalysts for biotechnology, medicine and bioremediation

Kumar

2010

Expert Opinion on Drug Metabolism & Toxicology

147

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“…This promiscuity provides opportunistic “starting points” for both in vivo and in vitro evolution of new functions. Indeed, the enhancement and exploitation of catalytic promiscuity has emerged as an important strategy for developing novel biocatalysts that has been reviewed extensively [5,6,14–18]. …”

Section: Natural Evolution Of Function: Mechanistically Diverse Supermentioning

confidence: 99%

“…Also, examples of computational (re)design of novel activities in unrelated structural scaffolds are described. Structure-based approaches for (re)design [e.g., 1–4] have been reviewed [e.g., 5,6]. …”

Section: Introductionmentioning

confidence: 99%

Enzyme (re)design: lessons from natural evolution and computation

Gerlt

Babbitt²

2009

Current Opinion in Chemical Biology

133

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Summary The (re)design of enzymes to catalyze “new” reactions is a topic of considerable practical and intellectual interest. Directed evolution (random mutagenesis followed by screening/selection) has been used widely to identify novel biocatalysts. However, “rational” approaches using either natural divergent evolution or computational predictions based on chemical principles have been less successful. This review summarizes recent progress in evolution- and computation-based (re)design.

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“…One promising area is computational protein design, which offers the possibility of creating enzymes for both natural and non-natural chemical transformations. 118−121 Although the enzymes designed to date generally possess activities that are low compared to those of natural enzymes, 118,122 these scaffolds serve as attractive starting points for further directed evolution 123−125 that could lead to optimized performance in vitro and in vivo . Bioinformatics methods that facilitate the organization of enzymes into superfamilies based on common tertiary structure and a fully or partially shared mechanism have also emerged.…”

Section: Future Perspectivesmentioning

confidence: 99%

Enzyme Recruitment and Its Role in Metabolic Expansion

Schulenburg

Miller

2014

Biochemistry

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Although more than 109 years have passed since the existence of the last universal common ancestor, proteins have yet to reach the limits of divergence. As a result, metabolic complexity is ever expanding. Identifying and understanding the mechanisms that drive and limit the divergence of protein sequence space impact not only evolutionary biologists investigating molecular evolution but also synthetic biologists seeking to design useful catalysts and engineer novel metabolic pathways. Investigations over the past 50 years indicate that the recruitment of enzymes for new functions is a key event in the acquisition of new metabolic capacity. In this review, we outline the genetic mechanisms that enable recruitment and summarize the present state of knowledge regarding the functional characteristics of extant catalysts that facilitate recruitment. We also highlight recent examples of enzyme recruitment, both from the historical record provided by phylogenetics and from enzyme evolution experiments. We conclude with a look to the future, which promises fruitful consequences from the convergence of molecular evolutionary theory, laboratory-directed evolution, and synthetic biology.

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Novel Enzymes Through Design and Evolution

Cited by 9 publications

References 148 publications

Engineering cytochrome P450 biocatalysts for biotechnology, medicine and bioremediation

Engineering cytochrome P450 biocatalysts for biotechnology, medicine and bioremediation

Enzyme (re)design: lessons from natural evolution and computation

Enzyme Recruitment and Its Role in Metabolic Expansion

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