# Exploring the Mutated Kinases for Chemoenzymatic Synthesis of N4-Modified Cytidine Monophosphates

## TL;DR

This paper describes a method to create modified cytidine monophosphates using chemical and enzymatic steps, with mutated enzymes enabling broader substrate use.

## Contribution

The study introduces mutated kinases that expand the range of substrates for enzymatic phosphorylation of modified cytidines.

## Key findings

- Mutant kinases phosphorylated N4-modified cytidines that wild-type enzymes could not.
- Point mutations in active sites significantly altered kinase substrate specificity.
- Chemoenzymatic synthesis successfully produced modified cytidine monophosphates.

## Abstract

Nucleosides, nucleotides, and their analogues are an important class of molecules that are used as substrates in research of enzymes and nucleic acid, or as antiviral and antineoplastic agents. Nucleoside phosphorylation is usually achieved with chemical methods; however, enzymatic phosphorylation is a viable alternative. Here, we present a chemoenzymatic synthesis of modified cytidine monophosphates, where a chemical synthesis of novel N4-modified cytidines is followed by an enzymatic phosphorylation of the nucleosides by nucleoside kinases. To enlarge the substrate scope, multiple mutant variants of Drosophila melanogaster deoxynucleoside kinase (DmdNK) (EC:2.7.1.145) and Bacillus subtilis deoxycytidine kinase (BsdCK) (EC:2.7.1.74) have been created and tested. It has been determined that certain point mutations in the active sites of the kinases alter their substrate specificities noticeably and allow phosphorylation of compounds that had been otherwise not phosphorylated by the wild-type DmdNK or BsdCK.