Abstract
Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data.
Highlights
Carbohydrates exist in many forms in nature, from the O- and N-linked glycans of proteins to the cell walls of plants, fungi and bacteria as well as storage polymers such as starch and glycogen
As was done in this study, may help in this matter as catalysis might limit the contribution of the active site to the observed binding with these techniques
Competition with small molecules known to bind at the active site, may help identify if binding is taking place elsewhere, caution is needed as these may bind at a potential surface binding sites (SBSs) or carbohydrate binding modules (CBMs)
Summary
Carbohydrates exist in many forms in nature, from the O- and N-linked glycans of proteins to the cell walls of plants, fungi and bacteria as well as storage polymers such as starch and glycogen. Enzymes active towards these carbohydrates must have the means to identify and act on their particular substrate within an often complex milieu. A common strategy for accomplishing this recognition is to utilize binding regions outside of the active site that are less constrained as they need not bind the substrate in an optimal mode for catalysis. Identifying Novel Carbohydrate Binding Sites in Enzymes funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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