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turns one nucleoside into another, via a sugar phosphate intermediate that never needs to be isolated (Figure 48).

This study describes how screening [4] of a panel of promiscuous wild-type pyrimidine nucleoside phosphorylases identified a candidate for phosphorolysis and transglycosylation reactions with nucleoside analogues carrying an alkylated sugar scaffold. The pyrimidine nucleoside phosphorylase from Thermus thermophilus (TtPyNP) is extremely stable [5] and selectively delivers an alkylated sugar phosphate, which can be transformed into a series of other nucleoside analogues. Experimental thermodynamic studies revealed that principles of thermodynamic control [3] could predict the maximum yields obtained in these reactions with striking accuracy, which proved empowering for further reaction optimisation. Remarkably, the wild-type TtPyNP already exhibited

synthetically useful activity without the need for further protein engineering, while all other tested nucleoside phosphorylases were inactive with the key substrate. Crystallographic data and docking studies suggest that a subtle threonine-serine substitution at the back of the active site enabled the enzyme to accommodate the sterically more demanding nucleoside analogue (Figure 49). In support of this hypothesis, introduction of this substitution in a previously inactive nucleoside phosphorylase (GtPyNP) installed a small but measurable activity.

Collectively, this research lays the foundation for the systematic exploration and characterisation of nucleoside phosphorolysis equilibrium systems and provides structural data and insights, which will facilitate the engineering of nucleoside phosphorylases for the diversification of nucleoside analogues.

Fig. 49: Docking of (a) the native substrate uridine (orange carbon atoms) and (b) the key methylated analogue 1a (purple carbon atoms) into the crystal structure of TtPyNP points to subtle steric changes in the back of the active site, which act as a gatekeeper for productive substrate binding. Docking of (c) uridine and (d) 1a into the crystal structure of the inactive enzyme GtPyNP shows that the sterically demanding nucleoside analogue preferably adapts an unproductive twisted conformation. The introduction of a threonine-serine substitution in the active site of the previously inactive GtPyNP resulted in a small but measurable activity.

PRINCIPAL PUBLICATION AND AUTHORS

Diversification of 4'-Methylated Nucleosides by Nucleoside Phosphorylases, F. Kaspar (a,b), M. Seeger (c), S. Westarp (a,b), C. Köllmann (d), A.P. Lehmann (d), P. Pausch (e), S. Kemper (f), P. Neubauer (a), G. Bange (e), A. Schallmey (c), D.B. Werz (d), A. Kurreck (a,b), ACS Catal. 11, 10830-10835 (2021); doi: https:/doi.org/10.1021/acscatal.1c02589 (a) Chair of Bioprocess Engineering, Technische Universität Berlin (Germany) (b) BioNukleo GmbH, Berlin (Germany) (c) Institute for Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig (Germany) (d) Institute of Organic Chemistry, Technische Universität Braunschweig (Germany) (e) Center for Synthetic Microbiology (SYNMIKRO), Philipps-University Marburg (Germany) (f) Institute for Chemistry, Technische Universität Berlin (Germany)

REFERENCES

[1] F. Kaspar et al., Green Chem. 23, 37-50 (2021). [2] F. Kaspar, R.T. Giessmann et al., Adv. Synth. Catal. 362, 867-876 (2020). [3] F. Kaspar, R.T. Giessmann et al., ChemBioChem 21, 1428-1432 (2020). [4] F. Kaspar et al., ChemBioChem 21, 2604-2610 (2020). [5] F. Kaspar et al., ChemBioChem 22, 1385-1390 (2021).