Improving the kinetic parameters of nicotine oxidizing enzymes by homologous structure comparison and rational design

Demand exists for a nicotine oxidase enzyme with high catalytic efficiency for a variety of applications including the in vivo detection of nicotine, therapeutic enzymatic blockade of nicotine from the CNS, and inactivation of toxic industrial wastes generated in the manufacture of tobacco products. Nicotine oxidase enzymes identified to date suffer from low efficiency, exhibiting either a high k_cat or low K_m, but not both. Here we present the crystal structure of the (S)-6-hydroxy-nicotine oxidase from Shinella sp HZN7 (NctB), an enzyme that oxidizes (S)-nicotine with a high k_cat (>1 s^-1), that possesses remarkable structural and sequence similarity to an enzyme with a nanomolar K_m for (S)-nicotine, the (S)-nicotine oxidase from Pseudomonas putidia strain S16 (NicA2). Based on a comparison of our NctB structure and the previously published crystal structure of NicA2, we successfully employed a rational design approach to increase the rate of oxidative turnover of the NicA2 enzyme by ∼25% (0.011 s^-1 to 0.014 s^-1), and reduce the K_m of the NctB protein by approximately 34% (940 μM-622 μM). While modest, these results are a step towards engineering a nicotine oxidase with kinetic parameters that fulfill the functional requirements of biosensing, waste remediation, and therapeutic applications.