PMID- 29057984 OWN - NLM STAT- MEDLINE DCOM- 20190712 LR - 20190712 IS - 2045-2322 (Electronic) IS - 2045-2322 (Linking) VI - 7 IP - 1 DP - 2017 Oct 20 TI - Membrane binding of the insertion sequence of Proteus vulgaris L-amino acid deaminase stabilizes protein structure and increases catalytic activity. PG - 13719 LID - 10.1038/s41598-017-14238-7 [doi] LID - 13719 AB - Proteus vulgaris L-amino acid deaminase (pvLAAD) belongs to a class of bacterial membrane-bound LAADs mainly express in genus Proteus, Providencia and Morganella. These LAADs employ a non-cleavable N-terminal twin-arginine translocation (Tat) peptide to transport across membrane and bind to bacterial surface. Recent studies revealed that a hydrophobic insertion sequence (INS) in these LAADs also interacts with bacterial membrane. However, the functional significance of INS-membrane interaction is not clear. In this study, we made site-directed mutagenesis on the surface-exposed hydrophobic residues of pvLAAD INS, and we found that these mutations impaired the INS-membrane interaction but did not affect pvLAAD activity in the solution. We further found that when cell membrane is present, the catalytic activity can be increased by 8~10 folds for wild-type but not INS-mutated pvLAAD, indicating that the INS-membrane interaction is necessary for increasing activity of pvLAAD. Molecular dynamic (MD) simulations suggested that INS is flexible in the solution, and its conformational dynamics could lead to substrate channel distortion. Circular dichroism (CD) spectroscopy experiments indicated that bacterial membrane was able to maintain the conformation of INS. Our study suggests the function of the membrane binding of INS is to stabilize pvLAAD structure and increase its catalytic activity. FAU - Ju, Yingchen AU - Ju Y AD - Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China. FAU - Liu, Zhihong AU - Liu Z AD - Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China. FAU - Zhang, Zizhen AU - Zhang Z AD - Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China. FAU - Duan, Lijun AU - Duan L AD - Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China. FAU - Liu, Qi AU - Liu Q AD - Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China. FAU - Gu, Qiong AU - Gu Q AD - Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China. FAU - Zhang, Cheng AU - Zhang C AD - Department of Pharmacology and Chemical Biology, School of Medicine, University of Pittsburgh, Pittsburgh, 15261, USA. FAU - Xu, Jun AU - Xu J AD - Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China. junxu@biochemomes.com. FAU - Zhou, Huihao AU - Zhou H AD - Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China. zhuihao@mail.sysu.edu.cn. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20171020 PL - England TA - Sci Rep JT - Scientific reports JID - 101563288 RN - 0 (Amino Acids) RN - 0 (Bacterial Proteins) RN - 0 (Liposomes) RN - 0 (Solutions) RN - EC 1.4.3.2 (L-Amino Acid Oxidase) SB - IM MH - Amino Acid Sequence MH - Amino Acids/metabolism MH - Bacterial Proteins/*chemistry/genetics/*metabolism MH - Catalysis MH - Cell Membrane/*metabolism MH - Circular Dichroism MH - Escherichia coli MH - Hydrophobic and Hydrophilic Interactions MH - Kinetics MH - L-Amino Acid Oxidase/*chemistry/genetics/*metabolism MH - Liposomes/metabolism MH - Molecular Dynamics Simulation MH - Mutagenesis, Site-Directed MH - Mutation MH - Protein Binding MH - Protein Conformation MH - Protein Stability MH - Proteus vulgaris/*enzymology MH - Solutions PMC - PMC5651824 COIS- The authors declare that they have no competing interests. EDAT- 2017/10/24 06:00 MHDA- 2019/07/13 06:00 PMCR- 2017/10/20 CRDT- 2017/10/24 06:00 PHST- 2017/07/17 00:00 [received] PHST- 2017/10/06 00:00 [accepted] PHST- 2017/10/24 06:00 [entrez] PHST- 2017/10/24 06:00 [pubmed] PHST- 2019/07/13 06:00 [medline] PHST- 2017/10/20 00:00 [pmc-release] AID - 10.1038/s41598-017-14238-7 [pii] AID - 14238 [pii] AID - 10.1038/s41598-017-14238-7 [doi] PST - epublish SO - Sci Rep. 2017 Oct 20;7(1):13719. doi: 10.1038/s41598-017-14238-7.