Abstract
In this study, we investigated MalS, a periplasmic α-enzyme from Escherichia coli K12, known for its unique biochemical properties related to polysaccharide utilization. Evolutionarily, MalS has inherited the glycosyl hydrolase catalytic domain from the glycoside hydrolase family 13, with the protein sequences highly conserved across Enterobacteria, including Salmonella and Shigella. MalS exhibited optimal activity at 65 °C, significantly higher than other E. coli enzymes. Although its reaction pattern resembled that of typical α-amylases, its catalytic efficiency on polysaccharides was notably lower. Intriguingly, MalS demonstrated a strong binding affinity for various glucose polymers, including β-cyclodextrin and glycogen, which significantly enhanced its thermostability. Despite full-length MalS binding strongly to glycogen, neither its N-terminal domain, predicted by AlphaFold2 to belong to the Carbohydrate-Binding Module family 69, nor the remaining parts of the enzyme showed binding affinity toward polysaccharides. Kinetic studies revealed that MalS had a 2.5-fold lower K(m) and 1.4-fold higher catalytic efficiency toward glycogen compared to amylopectin, which contrasts starkly with pancreatic α-amylases. However, over prolonged reactions, glycogen hydrolysis by MalS was slower than that of amylopectin. In the early initial stage, MalS predominantly degraded glycogen to maltopentaose (G5) rather than maltohexaose (G6) as usual. Taken together, these findings suggest MalS may play a role in recognizing glycogen-type polysaccharides in the bacterial periplasm during adaptation to new environments. Given the crucial role of glycogen in the survival and infection processes of pathogenic bacteria, understanding MalS's interaction with glycogen-type polysaccharides could offer valuable insights into bacterial survival mechanisms and their ability to infect hosts. KEY POINTS: • MalS has unique structure and properties but conserved among many enterobacteria • Binding of MalS with polysaccharides significantly enhanced its thermostability • Unlike other amylases, MalS showed 2.5-fold lower K(m) on glycogen than amylopectin.