2013 年 82 巻 1 号 p. 1_013-1_018
Cry39Aa from Bacillus thuringiensis serovar aizawai BUN1-14 is highly toxic to the larvae of Anopheles stephensi mosquitoes, which transmit malarial parasites. We constructed a homology model of Cry39Aa toxin using the reported structure of Cry4Ba toxin (PDB file 1W99) as a template, and we identified 349KYAYWR354 as the putative loop 1 in domain II. Many studies of various Cry toxins have shown that surface-exposed loops of domain II are critical for toxicity and receptor binding. To investigate the functional role of the putative loop 1 of Cry39Aa toxin, we performed site-directed mutagenesis in the loop. The results of alanine substitutions revealed that the entire structure of loop 1, and aromatic amino acids Y350 and Y352 in particular, are essential for larvicidal activity. In contrast, the toxicities of Cry39Aa mutants with phenylalanine substitutions at these two tyrosine residues did not differ markedly from the toxicity of wild-type Cry39Aa. A competitive binding assay involving A. stephensi brush border membrane vesicles revealed that the alanine mutants showed reduced competition with wild-type Cry39Aa toxin. Our results suggest that the molecular structure of loop 1 in domain II of Cry39Aa toxin is important for toxicity and receptor binding in the midgut of A. stephensi larvae.
