<i>In vivo</i> biosynthesis of tyrosine analogs and their concurrent incorporation into a residue-specific manner for enzyme engineering

Won, Yumi; Jeon, Hyeongtag; Pagar, Amol D.; Patil, Mahesh D.; Nadarajan, Sivakumar; Flood, Dillon T.; Dawson, Philip E.

doi:10.1039/c9cc08503c

Cited by 10 publications

(16 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, it is worth mentioning that the ( R )-ATA utilized herein was previously created from the DATA from Bacillus subtilis without affecting overall stability ( Voss et al, 2020 ). Incorporation of halogenated ncAAs by selective pressure incorporation or site-specific incorporation in several instances has enhanced the stability of enzymes ( Hoesl et al, 2011 ; Deepankumar et al, 2014 ; Carlsson et al, 2018 ; Ohtake et al, 2018 ; Won et al, 2019a ). Nevertheless, incorporation of single p BpA has not yet been reported for improved stability.…”

Section: Resultsmentioning

confidence: 99%

“…Autonomous biosynthesis of ncAAs and their concurrent incorporation into enzyme of interest in vivo could significantly reduce the production cost and permeability issues ( Pagar et al, 2021 ). Using enzymatic and metabolic pathways, some ncAAs like p -amino-phenylalanine, 5-hydroxytryptophan, l -phosphothreonine, l -dihydroxyphenylalanine, fluorotyrosine, and S -allylcysteine were biosynthesized in E. coli and concurrently incorporated into target proteins ( Ma et al, 2014 ; Exner et al, 2017 ; Kim et al, 2018 ; Won et al, 2019a ; Nojoumi et al, 2019 ; Schipp et al, 2020 ). The production of an increasing number of ncAAs in engineered cells will be aided by advances in biochemistry, molecular biology, and synthetic biology.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Non-Canonical Amino Acid-Based Engineering of (R)-Amine Transaminase

et al. 2022

Self Cite

View full text Add to dashboard Cite

Non-canonical amino acids (ncAAs) have been utilized as an invaluable tool for modulating the active site of the enzymes, probing the complex enzyme mechanisms, improving catalytic activity, and designing new to nature enzymes. Here, we report site-specific incorporation of p-benzoyl phenylalanine (pBpA) to engineer (R)-amine transaminase previously created from d-amino acid aminotransferase scaffold. Replacement of the single Phe88 residue at the active site with pBpA exhibits a significant 15-fold and 8-fold enhancement in activity for 1-phenylpropan-1-amine and benzaldehyde, respectively. Reshaping of the enzyme’s active site afforded an another variant F86A/F88pBpA, with 30% higher thermostability at 55°C without affecting parent enzyme activity. Moreover, various racemic amines were successfully resolved by transaminase variants into (S)-amines with excellent conversions (∼50%) and enantiomeric excess (>99%) using pyruvate as an amino acceptor. Additionally, kinetic resolution of the 1-phenylpropan-1-amine was performed using benzaldehyde as an amino acceptor, which is cheaper than pyruvate. Our results highlight the utility of ncAAs for designing enzymes with enhanced functionality beyond the limit of 20 canonical amino acids.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Non-Canonical Amino Acid-Based Engineering of (R)-Amine Transaminase

et al. 2022

Self Cite

View full text Add to dashboard Cite

show abstract

“…3C ). Similarly, other fluorinated l -tyrosine derivatives were also synthesized through TPL-catalyzed synthetic biological systems (Dennig et al 2015 ; Li et al 2020a ; Won et al 2019 ). Moreover, the donor substrates were generated in situ.…”

Section: Enzymatic Synthesis Of Fluorinated Compoundsmentioning

confidence: 99%

“…Therefore, the target compounds doped with fluorine are endowed with stronger activity and stability, longer half-life, and better bioabsorbability (Moschner et al 2019 ; Ni and Hu 2016 ; Reichel and Karaghiosoff 2020 ), especially in the fields of pharmaceutical intermediates (Gillis et al 2015 ; Swallow 2015 ; Zhdankin et al 2017 ; Zou et al 2019 ), cancer treatment (Meanwell et al 2020 ), antiviral agents (Pomeisl et al 2019 ), photovoltaics, diagnostic probes and bioinspired materials (Moschner et al 2019 ). Besides, natural proteins incorporating fluorinated amino acids showed also many unique characteristics, which are used in the biotherapeutics protein–protein interaction and the synthesis of high value-added chemicals (Arias et al 2020 ; Awad and Ayoup 2020 ; Bucci et al 2019 ; Liu et al 2019 ; Mei et al 2020a ; Remete et al 2018 ; Sisila et al 2021 ; Vaughan et al 2016 ; Wang and Matthews 2020 ; Won et al 2019 ).…”

Section: Introductionmentioning

confidence: 99%

Enzymatic synthesis of fluorinated compounds

Cheng

2021

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

Fluorinated compounds are widely used in the fields of molecular imaging, pharmaceuticals, and materials. Fluorinated natural products in nature are rare, and the introduction of fluorine atoms into organic compound molecules can give these compounds new functions and make them have better performance. Therefore, the synthesis of fluorides has attracted more and more attention from biologists and chemists. Even so, achieving selective fluorination is still a huge challenge under mild conditions. In this review, the research progress of enzymatic synthesis of fluorinated compounds is summarized since 2015, including cytochrome P450 enzymes, aldolases, fluoroacetyl coenzyme A thioesterases, lipases, transaminases, reductive aminases, purine nucleoside phosphorylases, polyketide synthases, fluoroacetate dehalogenases, tyrosine phenol-lyases, glycosidases, fluorinases, and multienzyme system. Of all enzyme-catalyzed synthesis methods, the direct formation of the C-F bond by fluorinase is the most effective and promising method. The structure and catalytic mechanism of fluorinase are introduced to understand fluorobiochemistry. Furthermore, the distribution, applications, and future development trends of fluorinated compounds are also outlined. Hopefully, this review will help researchers to understand the significance of enzymatic methods for the synthesis of fluorinated compounds and find or create excellent fluoride synthase in future research. Key points • Fluorinated compounds are distributed in plants and microorganisms, and are used in imaging, medicine, materials science . • Enzyme catalysis is essential for the synthesis of fluorinated compounds . • The loop structure of fluorinase is the key to forming the C-F bond . Supplementary Information The online version contains supplementary material available at 10.1007/s00253-021-11608-0.

show abstract

“…The reaction cleanly furnished the DNA-BSA conjugate (53) and ESI-TOF analysis of the crude reaction mixture indicated that no unmodified BSA remained in solution. 51,52 Conjugation to serum albumin is a valuable half-life increasing strategy that is commonly employed in the context of readily-cleared peptide and small protein drugs, and could in principle be applied to next-generation ASOs. [53][54][55][56][57] SENDR was also used to create a DNA construct that could be used in site-specific antibody conjugations ( Figure 6B).…”

Section: Sendr: Dna-protein Conjugatesmentioning

confidence: 99%

Synthetic Elaboration of Native DNA by RASS (SENDR)

Flood¹,

Knouse²,

Vantourout³

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

The controlled, site-specific ligation of molecules to native DNA remains an unanswered challenge. Herein, we report a simple solution to achieve this ligation through the tactical combination of two recently developed technologies: One for the manipulation of DNA in organic media, and another for the chemoselective labeling of alcohols. Reversible Adsorption of Solid Support (RASS) is employed to immobilize DNA and facilitate its transfer into dry acetonitrile. Subsequent ligation with P(V)-based Ψ reagents takes place in high yield with exquisite selectivity for the exposed 3’ or 5’ alcohols on DNA. This two-stage process, dubbed SENDR for Synthetic Elaboration of Native DNA by RASS, can be applied to a multitude of DNA conformations and sequences with a variety of functionalized Ψ reagents to generate useful constructs. Such entities can address numerous longstanding challenges, including the selective single coupling of DNA to proteins, ASOs, and functional small molecules, and also can allow the synthesis of doubly-labeled congeners for novel probe constructs including ones of potential interest to COVID-19 research. Finally, a prototype for the industrialization of SENDR in a kit format is presented.

show abstract

In vivo biosynthesis of tyrosine analogs and their concurrent incorporation into a residue-specific manner for enzyme engineering

Abstract: A cellular system for the in vivo biosynthesis of Tyr-analogs and their concurrent incorporation into target proteins is reported.

Cited by 10 publications

References 48 publications

Non-Canonical Amino Acid-Based Engineering of (R)-Amine Transaminase

Non-Canonical Amino Acid-Based Engineering of (R)-Amine Transaminase

Enzymatic synthesis of fluorinated compounds

Synthetic Elaboration of Native DNA by RASS (SENDR)

Contact Info

Product

Resources

About