Sutormin Oleg S, Nemtseva Elena V, Gulnov Dmitry V, Sukovatyi Lev A, Tyrtyshnaya Yekaterina S, Lisitsa Albert E, Kratasyuk Valentina A
Department of Chemistry, Institute of Natural and Technical Sciences, Surgut State University, Surgut, Russia.
Department of Biophysics, Institute of Fundamental Biology and Biotechnology, Siberian Federal University, Krasnoyarsk, Russia.
Photochem Photobiol. 2024 Mar-Apr;100(2):465-476. doi: 10.1111/php.13845. Epub 2023 Aug 15.
The study aims at revealing the mechanisms of the viscous medium effects on the kinetic features of NAD(P)H:FMN-oxidoreductase from luminous bacteria (Red), which are exhibited in a single enzyme assay and in coupling with bacterial luciferase (BLuc). Different concentrations of glycerol and sucrose were used to vary the medium viscosity. The activity of Red, alone and in the presence of BLuc, was analyzed, as well as BLuc activity in the presence of Red, whereas in the absence of BLuc, the Red activity was suppressed in viscous medium, and in the presence of BLuc, the increase in Red activity was observed at low glycerol concentrations (5-20 wt%). The interaction of glycerol and sucrose with Red substrates FMN and NADH was studied using absorption spectroscopy and molecular dynamics. Glycerol was found to form hydrogen bonds with the phosphate groups of the substrates, unlike sucrose. A mechanism for the activation of Red in the presence of BLuc in glycerol solutions through the acceleration of FMN reoxidation was proposed. Thus, it was concluded that, under the conditions used, the weakest link of the coupled enzyme system BLuc-Red in viscous medium is the FMN concentration, which depends on Red activity and the medium viscosity.
该研究旨在揭示粘性介质对发光细菌中NAD(P)H:FMN氧化还原酶（Red）动力学特性的影响机制，这些影响在单一酶分析以及与细菌荧光素酶（BLuc）偶联的分析中均有体现。使用不同浓度的甘油和蔗糖来改变介质粘度。分析了Red单独存在时以及在有BLuc存在时的活性，以及有Red存在时BLuc的活性，而在没有BLuc的情况下，Red活性在粘性介质中受到抑制，在有BLuc的情况下，在低甘油浓度（5 - 20 wt%）时观察到Red活性增加。使用吸收光谱和分子动力学研究了甘油和蔗糖与Red底物FMN和NADH的相互作用。发现甘油与底物的磷酸基团形成氢键，而蔗糖则不然。提出了在甘油溶液中通过加速FMN再氧化在有BLuc存在时激活Red的机制。因此，得出结论，在所使用的条件下，粘性介质中偶联酶系统BLuc - Red最薄弱的环节是FMN浓度，其取决于Red活性和介质粘度。
