Biochemical and Kinetic Characterization of the Glucose-6-Phosphate Dehydrogenase from Helicobacter pylori Strain 29CaP

Abstract

Helicobacter pylori (H. pylori) has been proposed as the foremost risk factor for the development of gastric cancer. We found that H. pylori express the enzyme glucose-6-phosphate dehydrogenase (HpG6PD), which participates in glucose metabolism via the pentose phosphate pathway. Thus, we hypothesized that if the biochemical and physicochemical characteristics of HpG6PD contrast with the host G6PD (human G6PD, HsG6PD), HpG6PD becomes a potential target for novel drugs against H. pylori. In this work, we characterized the biochemical properties of the HpG6PD from the H.pylori strain 29CaP and expressed the active recombinant protein, to analyze its steady-state kinetics, thermostability, and biophysical aspects. In addition, we analyzed the HpG6PD in silico structural properties to compare them with those of the HsG6PD. The optimal pH for enzyme activity was 7.5, with a T1/2 of 46.6 °C, at an optimum stability temperature of 37 °C. The apparent Km values calculated for G6P and NADP+ were 75.0 and 12.8 µM, respectively. G6P does not protect HpG6PD from trypsin digestion, but NADP+ does protect the enzyme from trypsin and guanidine hydrochloride (Gdn-HCl). The biochemical characterization of HpG6PD contributes to knowledge regarding H. pylori metabolism and opens up the possibility of using this enzyme as a potential target for specific and efficient treatment against this pathogen; structural alignment indicates that the three-dimensional (3D) homodimer model of the G6PD protein from H. pylori is different from the 3D G6PD of Homo sapiens.

Enabling High Activation of Glucose‐6‐Phosphate Dehydrogenase Activity Through Liquid Condensate Formation and Compression

“…Upon temperature‐induced unfolding, the α‐helix content decreases and aggregation of the protein, detected at 1618 cm −1 and 1685 cm −1 , due to formation of intermolecular molecular β‐sheets takes place, i. e., unfolding is followed by aggregation of the protein at high temperatures. The transition mid‐point temperature, T m , is located at 50.0±1.1 °C, which is in the same range observed in other G6PDHs from other organisms [62,63] . Table SI 1 summarizes the value for thermal unfolding of each structural component, which are all located at around 50 °C, indicating high cooperativity of the process.…”

“…The transition mid-point temperature, T m , is located at 50.0 � 1.1 °C, which is in the same range observed in other G6PDHs from other organisms. [62,63] Table SI 1 summarizes the value for thermal unfolding of each structural component, which are all located at around 50 °C, indicating high cooperativity of the process. The temperature-induced process of protein unfolding and subsequent aggregation was found to be irreversible.…”