Structural and Functional Analysis of Pyrimidine Nucleoside Phosphorylases of the NP-I and NP-II Families in Complexes w

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CTURE OF MACROMOLECULAR COMPOUNDS

Structural and Functional Analysis of Pyrimidine Nucleoside Phosphorylases of the NP-I and NP-II Families in Complexes with 6-Methyluracil I. I. Prokofeva,*, A. A. Lashkova, A. G. Gabdulkhakova, V. V. Balaeva, A. S. Mironovb, C. Betzelc, and A. M. Mikhailova† aShubnikov

Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics,” Russian Academy of Sciences, Moscow, 119333 Russia b State Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow, 117545 Russia c University of Hamburg, Hamburg, 20148 Germany *e-mail: [email protected] Received May 17, 2017

Abstract—The structure of bacterial uridine phosphorylase (UPh) belonging to the NP-I family in complex with 6-methyluracil was determined for the first time at 1.17 Å resolution. The structural features of bacterial UPh from the bacterium Vibrio cholerae (VchUPh) responsible for selectivity toward 6-methyluracil acting as a pseudosubstrate were revealed. The repulsion between the hydrophilic hydroxyl group of the active-site residue Thr93 of VchUPh and the hydrophobic methyl group of 6-methyluracil prevents the oxygen atom O4' of the ribose moiety and the phosphate oxygen atom O3P of ribose 1-phosphate from forming hydrogen bonds with OG1_Thr93, which are essential for the enzymatic reaction. This, apparently, makes VchUPh inactive in the enzymatic synthesis of 6-methyluridine from 6-methyluracil. Hence, Thr93 is the residue, the modification of which will allow VchUPh to catalyze the biotechnologically important synthesis of 6-methyluridine from 6-methyluracil. Taking into account high structural homology of the functionally significant regions of bacterial UPhs, this conclusion is also true for other bacterial UPhs. It was demonstrated that bacterial thymidine phosphorylases of the NP-II family cannot bind 6-methyluracil in a proper conformation required for the catalysis because of a close contact between the 6-methyl group and Phe210. DOI: 10.1134/S1063774518030239

INTRODUCTION Derivatives of pyrimidine nucleosides and nitrogenous bases are widely used in modern pharmaceutical industry. In particular, 6-methyluracil and 6-methyluridine are employed as anabolic and immune-stimulatory agents and as auxiliary agents for the antibiotic treatment of infectious diseases [1–6]. Besides, 6methyluracil (6MU) can be used to synthesize 6methyluridine and its derivatives (e.g., 5-fluoro-6hydroxymethyluridine), which are, in turn, promising antimetabolites for the treatment of cancer [7]. Uridine phosphorylase (UPh) is the enzyme that catalyzes the synthesis of thymidine or 5-methyluridine from thymine, as well as their phosphorolysis [8– 11]. However, it was demonstrated [12] that 6-methyluridine is not synthesized from 6MU by natural bacterial UPhs. Only the reverse phosphorolysis of 6methyluridine to 6-methyluracil takes place. Krajewska and Shugar [12] hypothesized that the methyl group attached to the aromatic ring in the 6 position of † Deceased.

6MU might produce ste