Efficiency of RNA Hydrolysis by Binase from Bacillus pumilus : The Impact of Substrate Structure, Metal Ions, and Low Mo
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CTURAL-FUNCTIONAL ANALYSIS OF BIOPOLYMERS AND THEIR COMPLEXES UDC 577.151.42/45
Efficiency of RNA Hydrolysis by Binase from Bacillus pumilus: The Impact of Substrate Structure, Metal Ions, and Low Molecular Weight Nucleotide Compounds A. A. Kuznetsovaa, A. A. Akhmetgalievab, V. V. Ulyanovab, O. N. Ilinskayab, O. S. Fedorovaa, *, and N. A. Kuznetsova, ** a
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, Novosibirsk, 630090 Russia b Institute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, Kazan, 420008 Russia *е-mail: [email protected] **е-mail: [email protected] Received April 13, 2020; revised April 28, 2020; accepted April 28, 2020
Abstract—Binase is an extracellular guanyl-preferring ribonuclease from Bacillus pumilus. The main biological function of binase is RNA degradation with the formation of guanosine-2',3'-cyclic phosphate and its subsequent hydrolysis to 3'-phosphate. Extracellular RNases are believed to be key agents that affect the functional activity of the body, as they directly interact with epithelial and immune cells. The biological effects of the enzyme may consist of both direct RNA degradation, and the accumulation of 2',3'-cGMP in the human body. In this work, we have performed a comparative analysis of the cleavage efficiency of model RNA substrates, i.e., short hairpin structures that contain guanosine at various positions. It has been shown that the hydrolysis efficiency of the model RNA substrates depends on the position of guanosine. We have also demonstrated the influence of various divalent metal ions and low molecular weight nucleotide compounds on the binase-catalyzed endoribonucleolytic reaction. Keywords: ribonuclease, binase, pre-steady-state kinetics, fluorescence DOI: 10.1134/S0026893320050064
INTRODUCTION Binase (Bi), an extracellular guanyl-specific ribonuclease from Bacillus pumilus [1], belongs to the N1/T1/U2 RNase family (IPR001887). The enzyme catalyzes hydrolysis of the phosphodiester bond between guanosine-3'-phosphate and the 5'-OH group of the neighboring nucleotide to form 2',3'-guanosine cyclophosphate, followed by hydrolysis of the latter to 3'-phosphate [2]. Although the main site of the RNA cleavage by binase is guanosine, the enzyme can also hydrolyze RNA at adenosine residues, but with lower efficiency. It has been shown [3, 4] that hydrolysis of the 2',3'-cyclophosphate intermediate is significantly slower than its formation, which leads to accumulation of 2',3'-cGMP and 2',3'-cAMP in the case of successively arranged purine nucleotides in RNA. The biological effects of binase, such as antitumor and antiviral activity [5‒7], may be due to both the direct degradation of RNA and the accumulation of 2',3'-cGMP in the human body. It should be noted that the biological role of 2',3'-cyclic derivatives has not yet been studied in detail but there are already data on their participation in the protection of cells against
damage and infection [8‒10]. It can be assum
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