The enzyme 6-Hydroxy-l-Nicotine oxidase (HLNO) is a flavin-dependent enzyme that catalyzes the first step in the pyridine pathway of oxidation of nicotine as a source of energy and nitrogen in several bacteria. Recombinant Arthrobacter nicotinovorans HLNO also catalyzes oxidation of (s)-nicotine at a low but measurable rate (Fitzpatrick et al., 2016, Biochemistry 55, 697–703). Rational design and bioinformatics approaches, based on the known high-resolution structure of this enzyme (RCSB: 3NG7), were employed to further enhance the catalytic turnover and stability of the enzyme using (S)-nicotine as substrate. The active site residue Tyr311 forms a hydrogen bond with the hydroxyl group of (S)-6-OH-nicotine within the catalytic pocket. Its replacement by a tryptophan residue reduced the k_cat for (S)-6-OH-nicotine by more than 6-fold and increased ~1.5-fold. Combining this mutation with two surface mutations that were predicted to enhance enzyme stability, further increased the k_cat for nicotine resulting in a comparatively robust oxidation of (s)-nicotine (k_cat >1 s⁻¹) at 37 °C, at the same time reducing the specificity for (S)–OH-nicotine (k_cat/K_M) by more than 100-fold and increasing that for (S)-nicotine by more than 2-fold. Interestingly, adding a maltose-binding protein (MBP) tag onto the N-terminus of HLNO markedly increased the thermal stability of the enzyme, extending the half-life at 37 °C from ~2 h to ~22 h. This effect was due almost entirely to increased FAD retention, an observation that may prove useful to improve flavin retention in other flavin-dependent monoamine oxidases.

酶 6- Hydroxy- l - Nicotine oxidase (HLNO) 是一种黄素依赖性酶，它催化吡啶途径中尼古丁氧化的第一步，尼古丁是几种细菌的能量和氮源。重组食烟节杆菌HLNO 还以低但可测量的速率催化 (s)-尼古丁的氧化（Fitzpatrick 等人，2016，Biochemistry 55, 697–703）。基于这种酶 (RCSB: 3NG7) 的已知高分辨率结构，合理设计和生物信息学方法被用于进一步提高酶的催化周转率和稳定性，使用 (S)-尼古丁作为底物。活性位点残基 Tyr311 与催化袋内的 (S)-6-OH-尼古丁的羟基形成氢键。它被色氨酸残基取代，将(S)-6-OH-尼古丁的k _cat降低了6 倍以上，增加了约 1.5 倍。将该突变与预计会增强酶稳定性的两个表面突变相结合，进一步增加了k _cat对于尼古丁，在 37 °C 下导致 (s)-尼古丁 ( k _cat >1 s ^-1 )的氧化反应比较强烈，同时降低了 (S)-OH-尼古丁的特异性 ( k _cat / K _M )超过 100 倍，并将 (S)-尼古丁的增加超过 2 倍。有趣的是，在 HLNO 的 N 末端添加麦芽糖结合蛋白 (MBP) 标签显着提高了酶的热稳定性，将 37 °C 下的半衰期从 ~2 小时延长至 ~22 小时。这种效果几乎完全是由于 FAD 保留增加，这一观察结果可能证明有助于改善其他黄素依赖性单胺氧化酶中的黄素保留。