Manipulating the substrate specificity of murine dihydrofolate reductase enzyme using an expanded set of amino acids

Zheng, Shaoyong; Lim, Sung In; Kwon, Inchan

doi:10.1016/j.bej.2015.03.011

Cited by 6 publications

(10 citation statements)

References 66 publications

(115 reference statements)

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“…More recently, the site‐specific incorporation of UAA 8 into murine dihydrofolate reductase (mDHFR) resulted in a 7.6‐fold enhancement of catalytic activity toward folate substrate. The lower K m value of 9.5 for the engineered enzyme clearly showed an increase in binding affinity toward folate, whereas the parent enzyme exhibited a K m value of 22.5 [77]. Likewise, substituting the key Phe residue of mDHFR with UAA 8 and 9 leads to increased binding affinity toward the dihydrofolate substrate up to 4.0 and 5.8‐fold respectively.…”

Section: Applications Of Enzyme Engineering Using Uaasmentioning

confidence: 99%

Incorporating unnatural amino acids to engineer biocatalysts for industrial bioprocess applications

Ravikumar

Nadarajan

Yoo

et al. 2015

Biotechnology Journal

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The bioprocess engineering with biocatalysts broadly spans its development and actual application of enzymes in an industrial context. Recently, both the use of bioprocess engineering and the development and employment of enzyme engineering techniques have been increasing rapidly. Importantly, engineering techniques that incorporate unnatural amino acids (UAAs) in vivo has begun to produce enzymes with greater stability and altered catalytic properties. Despite the growth of this technique, its potential value in bioprocess applications remains to be fully exploited. In this review, we explore the methodologies involved in UAA incorporation as well as ways to synthesize these UAAs. In addition, we summarize recent efforts to increase the yield of UAA engineered proteins in Escherichia coli and also the application of this tool in enzyme engineering. Furthermore, this protein engineering tool based on the incorporation of UAA can be used to develop immobilized enzymes that are ideal for bioprocess applications. Considering the potential of this tool and by exploiting these engineered enzymes, we expect the field of bioprocess engineering to open up new opportunities for biocatalysis in the near future.

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Section: Applications Of Enzyme Engineering Using Uaasmentioning

confidence: 99%

Incorporating unnatural amino acids to engineer biocatalysts for industrial bioprocess applications

Ravikumar

Nadarajan

Yoo

et al. 2015

Biotechnology Journal

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Section: Improvement Of Enzyme Activities By Site-specific Incorporationmentioning

confidence: 99%

“… a Reproduced with permission from ref . Copyright 2015 Elsevier. b mDHFR WT . c The docking score for FOL subtracted by the docking score for DHF. d The score was not obtained due to an calculation error. …”

Section: Improvement Of Enzyme Activities By Site-specific Incorporationmentioning

confidence: 99%

“… a Values ± regression standard error. b Reproduced with permission from ref . Copyright 2015 Elsevier. c Taken from ref . d ( k cat /K M for FOL)/( k cat /K M for DHF). e Relative to k cat /K M ratio of mDHFR WT . …”

Section: Improvement Of Enzyme Activities By Site-specific Incorporationmentioning

confidence: 99%

“…We hypothesized that expansion of the number of amino acids available for enzyme synthesis would allow design/screening of novel enzymes beyond the limits set by nature. Since general strategies of non-natural amino acid incorporation into enzymes were discussed in detail in other reviews, − herein we briefly explained the site-specific incorporation technique of non-natural amino acids into enzymes. Then, we focused on summarizing our contribution to two topics, forming enzyme complexes via bioconjugation and engineering of the enzyme active site using non-natural amino acids, which was not extensively handled in other reviews.…”

Section: Introductionmentioning

confidence: 99%

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Bioconjugation and Active Site Design of Enzymes Using Non-natural Amino Acids

Kwon

Yang

2017

Ind. Eng. Chem. Res.

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Enzymes are biocatalysts that play key roles in diverse chemical reactions in living organisms and industrial conversion processes generating value-added products. Since wild-type enzymes obtained from nature are normally not optimal for various applications, enzyme engineering is usually required for enhanced or new properties. Site-specific incorporation of a non-natural amino acid became a powerful protein-engineering tool. In this short review, we briefly summarize our contribution to enzyme complex formation and active site design of enzymes using the technique of site-specific incorporation of a nonnatural amino acid. First, site-specific incorporation of a non-natural amino acid at a permissive site of an enzyme led to bioconjugation to other molecules without compromising critical properties. Murine dihydrofolate reductase (mDHFR) was site-specifically conjugated to a biotin via click chemistry to achieve site-specific immobilization. Similarly, formate dehydrogenase (FDH) was site-specifically conjugated to an organometallic catalyst for cofactor regeneration. Furthermore, successive applications of two different click chemistries allowed site-specific coupling of FDH and mannitol dehydrogenase for the enhanced overall reaction efficiencies via substrate channeling effects and active site-orientation control. Besides enzyme bioconjugation, precise control of non-natural amino acid incorporation also allows for active site modification with a non-natural amino acid. Introduction of a bulky nonnatural amino acid into the mDHFR active site lowered binding affinity to its inhibitor methotrexate without compromising binding affinity to its substrate dihydrofolate. Similarly, introduction of a bulky non-natural amino acid into the mDHFR active site led to the alteration of substrate specificity toward a poor substrate folate over a good substrate dihydrofolate.

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Recent advancements in enzyme engineering via site-specific incorporation of unnatural amino acids

Zhu

Tang

Zheng

et al. 2021

World J Microbiol Biotechnol

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Manipulating the substrate specificity of murine dihydrofolate reductase enzyme using an expanded set of amino acids

Cited by 6 publications

References 66 publications

Incorporating unnatural amino acids to engineer biocatalysts for industrial bioprocess applications

Incorporating unnatural amino acids to engineer biocatalysts for industrial bioprocess applications

Bioconjugation and Active Site Design of Enzymes Using Non-natural Amino Acids

Recent advancements in enzyme engineering via site-specific incorporation of unnatural amino acids

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