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Publication Abstract from PubMed

Domain engineering, including domain truncation, fusion, or swapping, has become a common strategy to improve properties of enzymes, especially glycosyl hydrolases. However, there are few reports explaining the mechanism of increased activity from a protein structure perspective. Amy703 is an alkaline amylase with a unique N-terminal domain. Prior studies have shown that N-Amy, a mutant without an N-terminal domain, exhibits improved activity, stability, and calcium ion independence. In this study, we have used X-ray crystallography to determine the crystal structure of N-Amy and used AlphaFold2 to model the Amy703 structure, respectively. We further used size exclusion chromatography to show that Amy703 existed as a monomer, whereas N-Amy formed a unique dimer. It was found that the N-terminus of one monomer of N-Amy was inserted into the catalytic domain of its symmetrical subunit, resulting in the expansion of the catalytic pocket. This also significantly increased the pK(a) of the hydrogen donor Glu350, thereby enhancing substrate binding affinity and contributing to increased N-Amy activity. Meanwhile, two calcium ions were found to bind to N-Amy at different binding sites, which also contributed to the stability of protein. Therefore, this study provided new structural insights into the mechanisms of various glycosyl hydrolases.

N-terminal domain truncation yielded a unique dimer of polysaccharide hydrolase with enhanced enzymatic activity, stability and calcium ion independence.,Xiang L, Hu X, Du C, Wu L, Lu Z, Zhou J, Zhang G Int J Biol Macromol. 2024 May;266(Pt 2):131352. doi: , 10.1016/j.ijbiomac.2024.131352. Epub 2024 Apr 2. PMID:38574926^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.