The deletion of amino-terminal domain in Thermoactinomyces vulgaris R-47 alpha-amylases: Effects of domain N on activity, specificity, stability and dimerization

Thermoactinomyces vulgaris R-47 α-amylases, TVA I and TVA II, have a domain N, which is an extra structure in the family 13 enzymes. To investigate the roles of domain N in TVAs, we constructed TVAs-ΔN mutants which are deleted in domain N, and Y14, 16, 68A and Y41, 82, 95A mutants of TVA II. TVAs-ΔN were unstable under alkaline conditions, and their thermal stabilities were 10°C lower than that of wild-types. The specific activities of TVAs-ΔN for pullulan, starch, cyclodextrins, and oligosaccharides were drastically decreased, being about 1,500- to 10,000-fold smaller than those of wild-types. The k cat values of Y14, 16, 68A and Y41, 82, 95A for all tested substrates were markedly decreased, and the K m value of Y14, 16, 68A for α-CD and maltotriose were 25- and 3-fold larger, and that of Y41, 82, 92A for starch was 10-fold larger than that of the wild-type. TVA I and TVAs-ΔN in solution are a monomer, while TVA II is a homo-dimer, calculated by their molecular masses. These results suggest domain N in TVAs is an important structure for stabilization of enzymes, recognition and hydrolysis of substartes, and dimerization of TVA II.