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Biosynthesis of Nucleic Acids01:28

Biosynthesis of Nucleic Acids

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Nucleic acid biosynthesis is a fundamental biochemical process that produces the purine and pyrimidine nucleotides essential for DNA and RNA synthesis. This pathway maintains a balanced nucleotide pool, preventing imbalances that could jeopardize genetic integrity and cellular function. Given the crucial role of nucleotides, their synthesis is tightly regulated to ensure proper cellular homeostasis.Purine BiosynthesisThe biosynthesis of purine nucleotides begins with ribose-5-phosphate, a...
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One of the unique features of tRNA is the presence of modified bases. In some tRNAs, modified bases account for nearly 20% of the total bases in the molecule. Altogether, these unusual bases protect the tRNA from enzymatic degradation by RNases.
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RNA editing is a post-transcriptional modification where a precursor mRNA (pre-mRNA) nucleotide sequence is changed by base insertion, deletion, or modification. The extent of RNA editing varies from a few hundred bases, in mitochondrial DNA of trypanosomes, to a just single base, in nuclear genes of mammals. Even a single base change in the pre-mRNA can convert a codon for one amino acid into the codon for another amino acid or a stop codon. This type of re-coding can significantly affect the...
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Improved Nucleoside (2'-Deoxy)Ribosyltransferases Maximize Enzyme Promiscuity while Maintaining Catalytic Efficiency.

Peijun Tang1, Greice M Zickuhr2, Alison L Dickson2

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Engineered nucleoside 2'-deoxyribosyltransferase enzymes (NDTs) offer a biocatalytic solution for synthesizing diverse nucleoside analogues, overcoming complex chemical synthesis challenges for antiviral and anticancer drug development.

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Area of Science:

  • Biocatalysis
  • Enzyme Engineering
  • Medicinal Chemistry

Background:

  • Nucleoside analogues are vital therapeutics for infections and cancer.
  • Traditional chemical synthesis of nucleoside analogues is complex and inefficient.
  • Nucleoside 2 -deoxyribosyltransferase (NDT) enzymes show potential for biocatalysis.

Purpose of the Study:

  • To engineer NDT enzymes for improved catalytic efficiency with diverse nucleosides.
  • To expand the substrate scope of NDT enzymes for broader nucleoside analogue production.
  • To understand the molecular basis for enhanced enzyme activity and substrate specificity.

Main Methods:

  • Rational enzyme design and mutagenesis of NDTs from *Chroococcidiopsis thermalis* (CtNDT) and *Bacillus psychrosaccharolyticus* (BpNDT).
  • Determination of crystal structures of engineered enzyme variants.
  • Kinetic characterization of enzyme variants with various nucleoside substrates.

Main Results:

  • Engineered NDT variants exhibited enhanced catalytic efficiency towards ribonucleosides and 3 -deoxynucleosides.
  • Improvements in catalytic efficiency were primarily due to increased turnover rates.
  • Enzymes maintained broad nucleobase promiscuity, enabling the synthesis of over 100 distinct nucleoside products.
  • Crystal structures revealed molecular insights into the expanded substrate scope.

Conclusions:

  • Rational engineering of NDTs provides a robust biocatalytic platform for nucleoside analogue synthesis.
  • The developed enzymes and reaction conditions offer a versatile approach for generating diverse nucleoside products.
  • This strategy facilitates the development of novel therapeutics by simplifying access to complex nucleoside analogues.