In Vitro Reconstitution of the dTDP-l-Daunosamine Biosynthetic Pathway Provides Insights into Anthracycline Glycosylation

2021 ◽  
Author(s):  
F. Ifthiha Mohideen ◽  
Lan Huong Nguyen ◽  
Joël D. Richard ◽  
Sara Ouadhi ◽  
David H. Kwan
Keyword(s):  
Biosynthetic Pathway ◽  
In Vitro Reconstitution

In vitro reconstitution of the biosynthetic pathway of 3-hydroxypicolinic acid

2019 ◽  
Vol 17 (3) ◽  
pp. 454-460 ◽  
Author(s):  
Xuan Yun ◽  
Qian Zhang ◽  
Meinan Lv ◽  
Hai Deng ◽  
Zixin Deng ◽  
...  
Keyword(s):  
Natural Products ◽  
Building Block ◽  
Biosynthetic Pathway ◽  
In Vitro Reconstitution ◽  
Important Building Block

Four enzymes direct the biosynthesis of 3-hydroxypicolinic acid, an important building block of bacterial natural products.

scholarly journals Total In Vitro Biosynthesis of the Thioamitide Thioholgamide and Investigation of the Pathway

2022 ◽  
Author(s):  
Asfandyar Sikandar ◽  
Maria Lopatniuk ◽  
Andriy Luzhetskyy ◽  
Rolf Müller ◽  
Jesko Koehnke
Keyword(s):  
Biosynthetic Pathway ◽  
Protein Complexes ◽  
Downstream Processing ◽  
Leader Peptide ◽  
Oxidative Decarboxylation ◽  
Cancerous Cell ◽  
In Vitro Reconstitution ◽  
Modified Peptides ◽  
In Vitro Biosynthesis

Thioholgamides are ribosomally synthesized and post-translationally modified peptides (RiPPs) with potent activity against cancerous cell lines and an unprecedented structure. Despite being one of the most structurally and chemically complex RiPPs, very few biosynthetic steps have been elucidated. Here, we report the complete in vitro reconstitution of the biosynthetic pathway. We demonstrate that thioamidation is the first step and acts as a gatekeeper for downstream processing. Thr dehydration follows thioamidation, and our studies reveal that both these modifications require the formation of protein complexes – ThoH/I and ThoC/D. Harnessing the power of AlphaFold we deduce that ThoD acts as a lyase and also propose putative catalytic residues. ThoF catalyzes the oxidative decarboxylation of the terminal Cys and the subsequent macrocyclization is facilitated by ThoE. This is followed by Ser dehydration, which is also carried out by ThoC/D. ThoG is responsible for histidine bis-N-methylation, which is a prerequisite for His β-hydroxylation – a modification carried out by ThoJ. The last step of the pathway is the removal of the leader peptide by ThoK to afford mature thioholgamide.

scholarly journals In Vitro Reconstitution of a Bacterial Polysaccharide Biosynthetic Pathway

2010 ◽  
Vol 24 (S1) ◽  
Author(s):  
Guohui Zhao ◽  
Robert Woodward ◽  
Wen Yi ◽  
Lei Li ◽  
Hironobu Eguchi ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Bacterial Polysaccharide ◽  
In Vitro Reconstitution

scholarly journals Genomic identification and in vitro reconstitution of a complete biosynthetic pathway for the osmolyte di-myo-inositol-phosphate

2007 ◽  
Vol 104 (11) ◽  
pp. 4279-4284 ◽  
Author(s):  
D. A. Rodionov ◽  
O. V. Kurnasov ◽  
B. Stec ◽  
Y. Wang ◽  
M. F. Roberts ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Inositol Phosphate ◽  
In Vitro Reconstitution

scholarly journals In vitro reconstitution of indolmycin biosynthesis reveals the molecular basis of oxazolinone assembly

2015 ◽  
Vol 112 (9) ◽  
pp. 2717-2722 ◽  
Author(s):  
Yi-Ling Du ◽  
Lona M. Alkhalaf ◽  
Katherine S. Ryan
Keyword(s):  
Molecular Basis ◽  
Biosynthetic Pathway ◽  
Therapeutic Agents ◽  
Trna Synthetase ◽  
Ring Assembly ◽  
Coa Ligase ◽  
Biochemical Assays ◽  
In Vitro Reconstitution

The bacterial tryptophanyl–tRNA synthetase inhibitor indolmycin features a unique oxazolinone heterocycle whose biogenetic origins have remained obscure for over 50 years. Here we identify and characterize the indolmycin biosynthetic pathway, using systematic in vivo gene inactivation, in vitro biochemical assays, and total enzymatic synthesis. Our work reveals that a phenylacetate–CoA ligase-like enzyme Ind3 catalyzes an unusual ATP-dependent condensation of indolmycenic acid and dehydroarginine, driving oxazolinone ring assembly. We find that Ind6, which also has chaperone-like properties, acts as a gatekeeper to direct the outcome of this reaction. With Ind6 present, the normal pathway ensues. Without Ind6, the pathway derails to an unusual shunt product. Our work reveals the complete pathway for indolmycin formation and sets the stage for using genetic and chemoenzymatic methods to generate indolmycin derivatives as potential therapeutic agents.

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