A One-Pot Biocatalytic Cascade to Access Diverse L-Phenylalanine Derivatives from Aldehydes or Carboxylic Acids DOI Open Access
Shelby R. Anderson,

Madan R. Gopal,

Abigail P. Spangler

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 7, 2024

Abstract Non-standard amino acids (nsAAs) that are L-phenylalanine derivatives with aryl ring functionalization have long been harnessed in natural product synthesis, therapeutic peptide and diverse applications of genetic code expansion. Yet, to date these chiral molecules often the products poorly enantioselective environmentally harsh organic synthesis routes. Here, we reveal broad specificity multiple pyridoxal 5’-phosphate (PLP)-dependent enzymes, specifically an L-threonine transaldolase, a phenylserine dehydratase, aminotransferase, towards substrates contain side chains substitutions. We exploit this tolerance construct one-pot biocatalytic cascade achieves high-yield 18 from aldehydes under mild aqueous reaction conditions. demonstrate addition carboxylic acid reductase module enable biosynthesis may be less expensive or reactive than corresponding aldehydes. Finally, investigate scalability by developing lysate-based route for preparative-scale 4-formyl-L-phenylalanine, nsAA bio-orthogonal handle is not readily market-accessible. Overall, work offers efficient, versatile, scalable potential lower manufacturing cost democratize many valuable nsAAs.

Language: Английский

Directed Evolution and Unusual Protonation Mechanism of Pyridoxal Radical C–C Coupling Enzymes for the Enantiodivergent Photobiocatalytic Synthesis of Noncanonical Amino Acids DOI
Lei Cheng,

Zhiyu Bo,

Benjamin Krohn-Hansen

et al.

Journal of the American Chemical Society, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 23, 2025

Visible light-driven pyridoxal radical biocatalysis has emerged as a new strategy for the stereoselective synthesis of valuable noncanonical amino acids in protecting-group-free fashion. In our previously developed dehydroxylative C–C coupling using engineered PLP-dependent tryptophan synthases, an enzyme-controlled unusual α-stereochemistry reversal and pH-controlled enantiopreference were observed. Herein, through high-throughput photobiocatalysis, we evolved set stereochemically complementary PLP enzymes, allowing both l- d-amino with enhanced enantiocontrol across broad pH window. These newly acid synthases permitted use range organoboron substrates, including boronates, trifluoroborates, boronic acids, excellent efficiency. Mechanistic studies unveiled unexpected racemase activity earlier enzyme variants. This promiscuous was abolished shedding light on origin enantiocontrol. Further mechanistic investigations suggest switch proton donor to account stereoinvertive formation highlighting stereoinversion mechanism that is rare conventional two-electron enzymology.

Language: Английский

Citations

2
Expanding the genetic code: In vivo approaches for incorporating non-proteinogenic monomers DOI
Dongheon Lee,

S Yun,

Jong‐il Choi

et al.

The Journal of Microbiology, Journal Year: 2025, Volume and Issue: 63(3), P. e2501005 - e2501005

Published: March 28, 2025

Language: Английский

Citations

1
Long-Range Electrostatics in Serine Proteases: Machine Learning-Driven Reaction Sampling Yields Insights for Enzyme Design DOI Creative Commons
Alexander Zlobin, Valentina Maslova,

Julia Beliaeva

et al.

Journal of Chemical Information and Modeling, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 10, 2025

Computational enzyme design is a promising technique for producing novel enzymes industrial and clinical needs. A key challenge that this faces to consistently achieve the desired activity. Fundamental studies of natural revealed critical contributions from second-shell - even more distant residues their remarkable efficiency. In particular, such organize internal electrostatic field promote reaction. Engineering fields computationally proved be strategy, which, however, has some limitations. Charged necessarily form specific patterns local interactions may exploited structural integrity. As result, it impossible probe alone by substituting amino acids. We hypothesize an approach isolates influences residues' charges other could yield deeper insights. use molecular modeling with AI-enhanced QM/MM reaction sampling implement apply model serine protease subtilisin. find negative charge 8 Å away catalytic site crucial achieving enzyme's efficiency, contributing than 2 kcal/mol lowering barrier. contrast, positive second-closest charged residue opposes efficiency raising barrier 0.8 kcal/mol. This result invites discussion into role trade-offs might have taken place in evolution enzymes. Our transferable can help investigate preorganization believe study engineering direction advance both fundamental applied enzymology lead new powerful biocatalysts.

Language: Английский

Citations

0
Drug delivery for platinum therapeutics DOI
Hui Liu, Jiahui Zou, Xiaotong Li

et al.

Journal of Controlled Release, Journal Year: 2025, Volume and Issue: 380, P. 503 - 523

Published: Feb. 11, 2025

Language: Английский

Citations

0
19F NMR study of proteins with parallel incorporation of multiple fluorinated aromatic amino acids DOI Creative Commons
Manman Li,

Guohua Xu,

Qiong Wu

et al.

Magnetic Resonance Letters, Journal Year: 2025, Volume and Issue: unknown, P. 200193 - 200193

Published: Feb. 1, 2025

Language: Английский

Citations

0
Histidine Tag-Specific PEGylation Improves the Circulating Half-Life of TIMP2 DOI

Jack Toor,

Wiktoria Roksana Grabowska,

Adam L. Johnson

et al.

ACS Applied Bio Materials, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 21, 2025

An overarching limitation of therapeutic biologics is the limited half-life these proteins often exhibit once in circulation. PEGylation, chemical conjugation to poly(ethylene glycol) (PEG), a common strategy improve protein pharmacokinetics (PK) by enhancing stability, reducing immunogenicity, and decreasing renal clearance. Tissue Inhibitor Metalloproteinases 2 (TIMP2) 22 kDa matrisome that exhibits potential across range human disease models yet possesses short serum half-life. To advance development recombinant His-tagged TIMP2 (TIMP2), we utilized primary amine (1 kDa) site-specific histidine (10 its circulating Primary PEG molecules (TIMP2-a-PEG(n)) efficient, it produces multiple positional isomers are difficult purify. Furthermore, high levels can affect MMP-inhibitory activity TIMP2. Despite this, TIMP2-a-PEG(n) displays significant improvement (11.5-fold) versus unconjugated In contrast, targets tag, enabling purification mono-PEGylated (TIMP2-H-PEG(1)) di-PEGylated (TIMP2-H-PEG(2)) forms. Our findings demonstrate TIMP2-H-PEG(1) improved PK with enhanced stability 6.2-fold increase while maintaining activity. These results suggest PEGylation at C-terminal His6 tag promising approach for further preclinical as biologic.

Language: Английский

Citations

0
From lead to market: chemical approaches to transform peptides into therapeutics DOI
Caitlin L. Gare, Andrew M. White, Lara R. Malins

et al.

Trends in Biochemical Sciences, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 1, 2025

Language: Английский

Citations

0
Site‐Selectively Accelerating the Generation of β‐Linked Residue Isoaspartate in Proteins DOI Open Access
Qinghua Wu,

Xiaochen Yang,

Ying Wang

et al.

Angewandte Chemie International Edition, Journal Year: 2025, Volume and Issue: unknown

Published: March 11, 2025

Abstract Isoaspartate (isoAsp) is a β‐linked residue in proteins spontaneously generated through Asn deamidation or Asp dehydration and significantly affects protein properties. However, the sluggish site‐nonselective generation of isoAsp residues severely impedes in‐depth biological investigations as well exploitation its unique β‐linkage features. Herein, we introduce method that allows site‐selective rapid proteins. This leverages genetic incorporation side‐chain‐esterified derivative (BnD), which undergoes facile intramolecular arrangement to form key intermediate, aspartyl succinimide (Suc); subsequent hydrolysis Suc gives rise major product. On native sites proteins, including Cu/Zn superoxide dismutase calmodulin, demonstrate BnD‐mediated formation faster than generally by three orders magnitude.

Language: Английский

Citations

0
Site‐Selectively Accelerating the Generation of β‐Linked Residue Isoaspartate in Proteins DOI Open Access
Qifan Wu,

Xiaochen Yang,

Ying Wang

et al.

Angewandte Chemie, Journal Year: 2025, Volume and Issue: unknown

Published: March 11, 2025

Abstract Isoaspartate (isoAsp) is a β‐linked residue in proteins spontaneously generated through Asn deamidation or Asp dehydration and significantly affects protein properties. However, the sluggish site‐nonselective generation of isoAsp residues severely impedes in‐depth biological investigations as well exploitation its unique β‐linkage features. Herein, we introduce method that allows site‐selective rapid proteins. This leverages genetic incorporation side‐chain‐esterified derivative (BnD), which undergoes facile intramolecular arrangement to form key intermediate, aspartyl succinimide (Suc); subsequent hydrolysis Suc gives rise major product. On native sites proteins, including Cu/Zn superoxide dismutase calmodulin, demonstrate BnD‐mediated formation faster than generally by three orders magnitude.

Language: Английский

Citations

0
Simultaneous measurement of multiple fluorine labelling effect on GB1 stability by 19F NMR DOI
Manman Li,

Guohua Xu,

Zhou Gong

et al.

Talanta, Journal Year: 2025, Volume and Issue: unknown, P. 127959 - 127959

Published: March 1, 2025

Language: Английский

Citations

0