Improved Nucleoside (2'-Deoxy)Ribosyltransferases Maximize Enzyme Promiscuity while Maintaining Catalytic Efficiency.

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.