Discovery of Intermediates of lacZ β-Galactosidase Catalyzed Hydrolysis Using dDNP NMR

Abstract

Using dissolution dynamic nuclear polarization, the sensitivity of single scan solution state C NMR can be improved up to 4 orders of magnitude. In this study, the enzyme lacZ β-galactosidase from Escherichia coli was subjected to hyperpolarized substrate, and previously unknown reaction intermediates were observed, including a 1,1-linked disaccharide. The enzyme is known for making 1,6-transglycosylation, producing products like allolactose, that are also substrates. To analyze the kinetics, a simple kinetic model was developed and used to determine relative transglycosylation and hydrolysis rates of each of the intermediates, and the novel transglycosylation intermediates were determined as better substrates than the 1,6-linked one, explaining their transient nature. These findings suggest that hydrolysis and transglycosylation might be more complex than previously described.

Harnessing13C-labeledmyo-inositol to interrogate inositol phosphate messengers by NMR

“…Considering that the PtdInsPs and InsPs are metabolically linked, concomitant analysis of both groups of molecules could uncover unprecedented information on the coordination of these two messenger networks. While NMR suffers from an innate low sensitivity, advances using hyperpolarization methods in recent years have greatly enhanced the signal of 13 C nuclei and could help to increase the sensitivity of our method in order to reliably detect inositol phosphates of low abundance, such as 1PP-InsP 5 and InsP 8 , or PtdInsPs via NMR 51,52…”

The relation between crystal structure and the occurrence of quantum-rotor-induced polarization

“…Therefore, hyperpolarization methods are presently a hot topic (Halse, 2016;Köckenberger and Matysik, 2010;Kovtunov et al, 2018;Wang et al, 2019). Examples of these techniques are dynamic nuclear polarization (Ardenkjaer-Larsen, 2016; Kjeldsen et al, 2018;Lilly Thankamony et al, 2017;Milani et al, 2015;Ni et al, 2013), spin-exchange optical pumping (Hollenbach et al, 2016;Meersmann and Brunner, 2015;Norquay et al, 2018;Walker, 2011), photochemically induced dynamic nuclear polarization (Bode et al, 2012;Kiryutin et al, 2012;Sosnovsky et al, 2019), and para-hydrogen-induced polarization (Duckett and Mewis, 2012;Kiryutin et al, 2017;Korchak et al, 2009). Another technique is quantum-rotorinduced polarization (QRIP; Dumez et al, 2017;Horsewill, 1999;Icker and Berger, 2012;Meier, 2018).…”

The relation between crystal structure and the occurrence of quantum-rotor induced polarization

“…Therefore, hyperpolarization methods are presently a "hot" issue (Halse, 2016;Köckenberger and Matysik, 2010;Kovtunov et al, 2018;Wang et al, 2019). Examples of these techniques are dynamic nuclear polarization (Ardenkjaer-Larsen, 2016;Kjeldsen et al, 2018;Lilly Thankamony et al, 2017;Milani et al, 2015;Ni et al, 2013), spin-exchange optical pumping (Hollenbach et al, 2016;Meersmann and Brunner, 2015;Norquay et al, 2018;Walker, 2011), photochemically induced dynamic nuclear polarization (Bode et al, 2013;Kiryutin et al, 2012;Sosnovsky et al, 2019) and para-hydrogen induced polarization (Duckett and Mewis, 2012;Kiryutin et al, 2017;Korchak et al, 2009). Another technique is quantum-rotor induced polarization (QRIP) (Dumez et al, 2017;Horsewill, 1999;Icker et al, 2013;Icker and Berger, 2012;Ludwig et al, 2010).…”