A molecular dynamics-guided mutagenesis identifies two aspartic acid residues involved in the pH-dependent activity of OG-OXIDASE 1

Berberine Bridge Enzyme-Like Oligosaccharide Oxidases Act As Enzymatic Transducers Between Microbial Glycoside Hydrolases and Plant Peroxidases

“…The constructs expressing Arabidopsis OGOX1 (pPICZαB/H-OGOX1) and the polygalacturonase from Fusarium phyllophilum (FpPG, pGAPZαA/FpPG) were the same used in (Benedetti et al, 2011; Scortica et al, 2021), respectively. The gene encoding the mature CELLOX from A. thaliana (AT4G20860) was fused downstream of the SUMOstar sequence developed by LifeSensor Inc. (https://lifesensors.com/) that also included the sequences encoding the FLAG-(DYKDDDDK) and 6xHistags(HHHHHH)(https://lifesensors.com/wpcontent/uploads/2019/09/2160_2161_Pichia_SUMOstar_Manual-1.pdf).…”

“…OGOX1 and FpPG were heterologously expressed in P. pastoris by following the same procedures described in (Benedetti et al, 2011; Scortica et al, 2021), respectively. Transformation and selection of Pichia transformants expressing FHS-CELLOX were performed by following the same procedures described in (Scortica et al, 2021) with some modifications.…”

“…The oxidized oligosaccharides are characterized by an increased recalcitrance to enzymatic hydrolysis (Benedetti et al, 2018), but nothing is known about their involvement in other physiological processes. Recently, the combined use of Arabidopsis OGOX1 and a peroxidase (POD) allowed the measurement of the OGOX1 activity suggesting that possible physiological processes could be driven by the OGOX-generated H 2 O 2 in the presence of POD (Scortica et al, 2021). In the present study, the capability of generating H 2 O 2 by combinations of OGOX1 with a microbial polygalacturonase and CELLOX with a microbial endoglucanase was tested.…”

“…FpPG, OGOX1 and CELLOX were expressed in P. pastoris and purified to homogeneity. The expression of OGOX1, was achieved as reported in (Scortica et al, 2021), whereas the expression of CELLOX, due to the low yield and high protein instability, required a different expression strategy that consisted in the addition of a Flag-6xHis-SUMOstar tag upstream of the sequence encoding CELLOX (Fig. S1A).…”

“…S1C). OGOX1 was purified from the culture filtrate of P. pastoris by two hydrophobic interaction chromatography steps performed at two different pH values (5.0 and 7.0) (Scortica et al, 2021), whereas FHS-CELLOX was purified in a single step by IMAC chromatography. The AnEG used in our experiments was a highly pure preparation from a commercial source whereas FpPG was constitutively expressed in P. pastoris and purified using a three-step purification procedure as reported in (Benedetti et al, 2011).…”