Substitution of one calcium-binding amino acid strengthens substrate binding in a thermophilic alginate lyase

Abstract

Ligand binding is sensitive to temperatures since noncovalent bonds between the binding site and ligand could be broken by heat. How metal ion-binding amino acids in alginate lyase evolve to achieve tight substrate binding in a hostile environment remains unknown. An endolytic alginate lyase AlgAT0 specifically cleaved the M-G glycosidic bond and released disaccharides as the main end product. Four conserved calcium-binding sites were predicted and the supplement of Ca²⁺ led to enhanced substrate binding and protein stability. Among the four conserved calcium-binding sites, one substitution of aspartate for glutamate in AlgAT0 was proved to stimulate Ca²⁺ affinity. This study suggested that substrate affinity of polysaccharide lyases could be improved by tight binding to Ca²⁺ via one amino acid substitution.

Keywords: Defluviitalea phaphyphila; Thermophile; alginate; calcium-binding sites; polysaccharide lyase.