Dynamic Expression of α-1,2-Glucan Synthase in Nostoc sp. PCC 7120 and Biochemical Characterization of an α-1,2-Glucan phosphorylase

Abstract

α-1,2-glucans are polysaccharides characterized by their unique α-1,2-glycosidic bonds and right-handed helical structure, which suggest novel biological functions. The alr1000 protein in Nostoc sp. PCC 7120 is the key enzyme for synthesizing α-1,2-glucans. In this study, we prepared rabbit antiserum for alr1000 and investigated its expressions when Nostoc sp. PCC 7120 is subjected to high salinity and high temperature stress. The results showed that under high salinity (up to 200 mM) and high temperature (40°C) conditions, alr1000 expression reached its highest level at 4 hours. This suggests that the elevated expression of alr1000 initiates massive production of α-1,2-glucans within Nostoc sp. PCC 7120, which helps the organism survive these stressful conditions. In addition, we identified and characterized the degrading enzymes of α-1,2-glucans. All4989, a GH65 family α-1,2-glucan phosphorylase from Nostoc sp. PCC 7120, was found to degrade α-1,2-glucans through a mechanism where a phosphate serves as the nucleophile to cleave the α-1,2-glycosidic bonds of α-1,2-glucans, while Glu485 functions as a general acid. Furthermore, the enzyme also catalyzes an efficient reverse reaction, synthesizing α-1,2-glucans of different molecular weights using breakdown products as substrates. Mechanistically, this process involves a key functional shift: Glu485 acts as a general acid during the forward degradation and switches to acting as a general base during the reverse synthesis.