title: The N-Terminal α-Helix Domain ofPseudomonas aeruginosaLipoxygenase Is Required for Its Soluble Expression inEscherichia colibut Not for Catalysis.
abstract: Lipoxygenase (LOX) is an industrial enzyme with wide applications in food and pharmaceutical industries. The available structure information indicates that eukaryotic LOXs consist of N terminus β-barrel and C terminus catalytic domains. However, the latest crystal structure ofPseudomonas aeruginosaLOX shows it is significantly different from those of eukaryotic LOXs, including the N-terminal helix domain. In this paper, the functions of this N-terminal helix domain in the soluble expression and catalysis ofP. aeruginosaLOX were analyzed. Genetic truncation of this helix domain resulted in an insolubleP. aeruginosaLOX mutant. The active C-terminal domain was obtained by dispase digestion of theP. aeruginosaLOX derivative containing the genetically introduced dispase recognition sites. This functional C-terminal domain showed raised substrate affinity but reduced catalytic activity and thermostability. Crystal structure analyses demonstrate that the broken polar contacts connecting the two domains and the exposed hydrophobic substrate binding pocket may contribute to the insoluble expression of the C terminus domain and the changes in the enzyme properties. Our data suggest that the N terminus domain ofP. aeruginosaLOX is required for its soluble expression inE. coli, which is different from that of the eukaryotic LOXs. Besides this, this N-terminal domain is not necessary for catalysis but shows positive effects on the enzyme properties. The results presented here provide new and valuable information on the functions of the N terminus helix domain ofP. aeruginosaLOX and further improvement of its enzyme properties by molecular modification.
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, P.R. China.
authors.forename: Xinyao
authors.initials: X
authors.lastname: Lu
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, P.R. China.
authors.forename: Guangsheng
authors.initials: G
authors.lastname: Wang
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, P.R. China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Feng
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, P.R. China.
authors.forename: Song
authors.initials: S
authors.lastname: Liu
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, P.R. China.
authors.forename: Xiaoman
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, P.R. China.|The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, P.R. China.
authors.forename: Guocheng
authors.initials: G
authors.lastname: Du
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, P.R. China.|National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, P.R. China.
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D008084:Lipoxygenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012995:Solubility
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D008084:Lipoxygenase
keywords: Lipoxygenase; N-terminal domain; Pseudomonas aeruginosa; catalysis; soluble expression