Tandem domains differentially affect enzyme activity in the sialidases of Glaesserella parasuis, Clostridium perfringens, and Bifidobacterium bifidum

Abstract Carbohydrate-active enzymes are often associated in tandem with various functional and structural domains, including hydrolytic enzymes, carbohydrate-binding modules, lectin domains, domains of unknown function, signal peptides, and immunoglobulin folds. Among these, carbohydrate-binding modules and lectin domains are well known to influence carbohydrate-active enzyme activity. Here, we investigated the impact of naturally occurring tandem domains on the hydrolytic activity of two well-characterized multi-domain sialidases: Bifidobacterium bifidum sialidase (BbSia2) and Glaesserella parasuis sialidase (HpNanH). We found that the sialidase activity of the BbSia2 catalytic domain remained unaffected upon deletion of its naturally occurring tandem domains. In contrast, deleting the naturally occurring tandem domains of HpNanH significantly reduced its sialidase activity. We also found that the non-native tandem placement of a previously reported sialic acid-binding module, MU3, enhanced the sialidase activity of the HpNanH catalytic domain, with the C-terminal positioning of MU3 providing a greater enhancement. However, the non-native tandem placement of MU3 or the non-catalytic tandem domains of HpNanH failed to enhance the sialidase activity of the catalytic domain of another well-characterized sialidase - Clostridium perfringens sialidase (CpNanI). These results highlight the complex and context-dependent roles of tandem domains in modulating carbohydrate-active enzyme activity and have implications for enzyme engineering studies.