Saltational evolution of a pesticide-metabolizing cytochrome P450 in a global crop pest

BACKGROUND The cotton bollworm, Helicoverpa armigera (Hubner), is a damaging insect pest threatening agricultural crops worldwide as a result of its resistance to insecticides. Metabolic resistance to pyrethroid insecticides is conferred by the chimeric P450 enzyme CYP337B3, produced by unequal crossing-over between CYP337B1 and CYP337B2. CYP337B3 is 99.7% similar to CYP337B1 except for the 177 N-terminal amino acids (AAs) containing the substrate recognition site 1 from CYP337B2. Here, we studied the structure-function relationship of CYP337B3 and CYP337B1 to determine the AAs that enable CYP337B3 to efficiently hydroxylate the 4 '-carbon position of fenvalerate, which neither CYP337B1 nor CYP337B2 can do. RESULTS Site-directed mutagenesis showed that the L114F substitution in CYP337B3 reduced its 4 '-hydroxylation activity by 89%, but the reciprocal F114L substitution in CYP337B1 increased its 4 '-hydroxylation activity to only 49% of the level of CYP337B3. Docking models showed that AA 114 seems to have different functions in CYP337B1 and CYP337B3. Antibodies detected two- to three-fold more CYP337B1 than CYP337B3 in larval cuticle, which along with a 49% 4 '-hydroxylation activity increase due to a F114L substitution in vivo might be expected to provide as much protection for the larva against exposure to fenvalerate as CYP337B3. However, CYP337B3 is present at much higher frequencies than CYP337B1-CYP337B2 in most populations, including those recently invading South America. CONCLUSION The metabolic resistance to pyrethroids in H. armigera has evolved by saltational evolution - by a single mutation, an unequal crossing-over, producing a larger selective advantage than could be attained gradually by stepwise improvement of the parental enzyme.

Automated summary

The metabolic resistance to pyrethroids in the cotton bollworm has evolved through a unique chimeric enzyme, CYP337B3, which efficiently hydroxylates fenvalerate at the 4'-carbon position. Site-directed mutagenesis and docking models revealed the specific amino acids and structural features that enable CYP337B3 to exhibit this unique enzymatic function.