An active site mutation in 6-hydroxy-l-Nicotine oxidase from Arthrobacter nicotinovorans changes the substrate specificity in favor of (S)-nicotine.
An active site mutation in 6-hydroxy-l-Nicotine oxidase from Arthrobacter nicotinovorans changes the substrate specificity in favor of (S)-nicotine.
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DOI：
10.1016/j.abb.2020.108520
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发表时间：
2020-08
影响因子：
3.9
通讯作者：
D. Deay;K. K. Colvert-K.;Fei Gao;S. Seibold;Priyanka Goyal;D. Aillon;P. Petillo;M. Richter
中科院分区：
文献类型：
--
作者：
D. Deay;K. K. Colvert-K.;Fei Gao;S. Seibold;Priyanka Goyal;D. Aillon;P. Petillo;M. Richter
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. RecombinantArthrobacternicotinovoransHLNO 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 thekcatfor (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 thekcatfor nicotine resulting in a comparatively robust oxidation of (s)-nicotine (kcat>1 s−1) at 37 °C, at the same time reducing the specificity for (S)–OH-nicotine (kcat/KM) 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.
