Structural and biochemical basis of a marine bacterial glycoside hydrolase family 2 β-glycosidase with broad substrate specificity

Uronic acids are commonly found in marine polysaccharides and increase structural complexity sanand intrinsic recalcitrance to enzymatic attack. The glycoside hydrolase family 2 (GH2) include proteins that target sugar conjugates with hexuronates and are involved in the catabolism and cycling of marine polysaccharides. Here, we reported a novel GH2, Aq GalA from a marine algae-associated Bacteroidetes with broad-substrate specificity. Biochemical analyses revealed that Aq GalA exhibits hydrolyzing activities against β-galacturonide, β-glucuronide, and β-galactopyranoside via retaining mechanisms. We solved the Aq GalA crystal structure in complex with galacturonic acid (GalA) and showed (via mutagenesis) that charge characteristics at uronate-binding subsites controlled substrate selectivity for uronide hydrolysis. Additionally, conformational flexibility of the Aq GalA active site pocket was proposed as a key component for broad substrate enzyme selectivity. Our Aq GalA structural and functional data augments the current understanding of substrate recognition of GH2 enzymes and provided key insights into the bacterial use of uronic acid containing polysaccharides. IMPORTANCE The decomposition of algal glycans driven by marine bacterial communities represents one of the largest heterotrophic transformation of organic matter fueling marine food webs and global carbon cycling. However, our knowledge of the carbohydrate cycling is limited due to structural complexity of marine polysaccharides and the complicated enzymatic machinery of marine microbes. To degrade algal glycan, marine bacteria such as members of Bacteroidetes produce a complex repertoire of carbohydrate-active enzymes (CAZymes) matching the structural specificity of the different carbohydrates. In this study, we investigated an extracellular GH2 β-glycosidase, Aq GalA from a marine Bacteroidetes to identify the key components responsible for glycuronides recognition and hydrolysis. The broad substrate specificity of Aq GalA against glycosides with diverse stereochemical substitutions indicates its potential in processing complex marine polysaccharides. Our findings promote a better understanding of microbially-driven mechanisms of marine carbohydrate cycling.