Molecular Deceleration Regulates Toxicant Release to Prevent Cell Damage in Pseudomonas putida S16 (DSM 28022) Hongzhi Tang, a,b,c Kunzhi Zhang, a,b,c,d Haiyang Hu, a,b,c Geng Wu, a,b,c Weiwei Wang, a,b,c Xiongyu Zhu, a,b,c Gongquan Liu, a,b,c Ping Xu a,b,c a State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai, People’s Republic of China b Joint International Research Laboratory of Metabolic and Developmental Sciences, Shanghai Jiao Tong University, Shanghai, People’s Republic of China c School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, People’s Republic of China d Zhejiang Center for Medical Device Evaluation, Zhejiang Medical Products Administration, Hangzhou, People’s Republic of China Hongzhi Tang, Kunzhi Zhang, Haiyang Hu, and Geng Wu contributed equally to the development of this article. Author order was determined by the temporal order in which the authors began performing the research. ABSTRACT The underlying molecular mechanisms of flavin-dependent amine oxidases remain relatively poorly understood, even though many of these en- zymes have been reported. The nicotine oxidoreductase NicA2 is a crucial en- zyme for the first step of nicotine degradation in Pseudomonas putida S16 (DSM 28022). Here, we present the crystal structure of a ternary complex comprising NicA2 residues 21 to 482, flavin adenine dinucleotide (FAD), and nicotine at 2.25 Å resolution. Unlike other, related structures, NicA2 does not have an associ- ated diacyl glycerophospholipid, wraps its substrate more tightly, and has an in- triguing exit passage in which nine bulky amino acid residues occlude the re- lease of its toxic product, pseudooxynicotine (PN). The replacement of these bulky residues by amino acids with small side chains effectively increases the catalytic turnover rate of NicA2. Our results indicate that the passage in wild- type NicA2 effectively controls the rate of PN release and thus prevents its rapid intracellular accumulation. It gives ample time for PN to be converted to less- harmful substances by downstream enzymes such as pseudooxynicotine amine oxidase (Pnao) before its accumulation causes cell damage or even death. The temporal metabolic regulation mode revealed in this study may shed light on the production of cytotoxic compounds. IMPORTANCE Flavin-dependent amine oxidases have received extensive attention because of their importance in drug metabolism, Parkinson’s disease, and neu- rotransmitter catabolism. However, the underlying molecular mechanisms remain rel- atively poorly understood. Here, combining the crystal structure of NicA2 (an en- zyme in the first step of the bacterial nicotine degradation pathway in Pseudomonas putida S16 (DSM 28022)), biochemical analysis, and mutant construction, we found an intriguing exit passage in which bulky amino acid residues occlude the release of the toxic product of NicA2, in contrast to other, related structures. The selective product exportation register for NicA2 has proven to be beneficial to cell growth. Those seeking to produce cytotoxic compounds could greatly benefit from the use of such an export register mechanism. KEYWORDS nicotine oxidoreductase, NicA2, pseudooxynicotine, crystal structure, molecular mechanism M icrobial cells are capable of ameliorating environmental exposures by metabo- lizing toxicants (1, 2). Examples include the use of bacteria to reduce toxicity at sites contaminated by polycyclic aromatic hydrocarbons (PAHs), dioxins, and nicotine Citation Tang H, Zhang K, Hu H, Wu G, Wang W, Zhu X, Liu G, Xu P. 2020. Molecular deceleration regulates toxicant release to prevent cell damage in Pseudomonas putida S16 (DSM 28022). mBio 11:e02012-20. https:// doi.org/10.1128/mBio.02012-20. Editor Sang Yup Lee, Korea Advanced Institute of Science and Technology Copyright © 2020 Tang et al. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Hongzhi Tang, tanghongzhi@sjtu.edu.cn, or Ping Xu, pingxu@sjtu.edu.cn. This article is a direct contribution from Ping Xu, a Fellow of the American Academy of Microbiology, who arranged for and secured reviews by Yaojun Tong, The Novo Nordisk Foundation Center for Biosustainability, and Pablo Ivan Nikel, Novo Nordisk Foundation Center for Biosustainability. Received 23 July 2020 Accepted 27 July 2020 Published RESEARCH ARTICLE Molecular Biology and Physiology crossm September/October 2020 Volume 11 Issue 5 e02012-20 ® mbio.asm.org 1 1 September 2020 Downloaded from https://journals.asm.org/journal/mbio on 24 February 2022 by 54.242.133.186.