title: Mutagenesis and redox partners analysis of the P450 fatty acid decarboxylase OleTJE.
abstract: The cytochrome P450 enzyme OleTJEfrom Jeotgalicoccus sp. ATCC 8456 is capable of converting free long-chain fatty acids into α-alkenes via one-step oxidative decarboxylation in presence of H2O2as cofactor or using redox partner systems. This enzyme has attracted much attention due to its intriguing but unclear catalytic mechanism and potential application in biofuel production. Here, we investigated the functionality of a select group of residues (Arg245, Cys365, His85, and Ile170) in the active site of OleTJEthrough extensive mutagenesis analysis. The key roles of these residues for catalytic activity and reaction type selectivity were identified. In addition, a range of heterologous redox partners were found to be able to efficiently support the decarboxylation activity of OleTJE. The best combination turned out to be SeFdx-6 (ferredoxin) from Synechococcus elongatus PCC 7942 and CgFdR-2 (ferredoxin reductase) from Corynebacterium glutamicum ATCC 13032, which gave the highest myristic acid conversion rate of 94.4%. Moreover, Michaelis-Menton kinetic parameters of OleTJEtowards myristic acid were determined.
authors.affiliation: Shandong Provincial Key Laboratory of Synthetic Biology, CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao 266101, China.|University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Fang
authors.affiliation: Shandong Provincial Key Laboratory of Synthetic Biology, CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao 266101, China.
authors.forename: Huifang
authors.initials: H
authors.lastname: Xu
authors.affiliation: Shandong Provincial Key Laboratory of Synthetic Biology, CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao 266101, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Shandong Provincial Key Laboratory of Synthetic Biology, CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao 266101, China.
authors.forename: Fengxia
authors.initials: F
authors.lastname: Qi
authors.affiliation: Shandong Provincial Key Laboratory of Synthetic Biology, CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao 266101, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Shandong Provincial Key Laboratory of Synthetic Biology, CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao 266101, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Chen
authors.affiliation: Shandong Provincial Key Laboratory of Synthetic Biology, CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao 266101, China.
authors.forename: Cong
authors.initials: C
authors.lastname: Wang
authors.affiliation: Shandong Provincial Key Laboratory of Synthetic Biology, CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao 266101, China.|University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Yilin
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Shandong Provincial Key Laboratory of Synthetic Biology, CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao 266101, China.
authors.forename: Wenxia
authors.initials: W
authors.lastname: Yang
authors.affiliation: Shandong Provincial Key Laboratory of Synthetic Biology, CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao 266101, China.
authors.forename: Shengying
authors.initials: S
authors.lastname: Li
mesh_terms: D000475:Alkenes / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003653:Decarboxylation; D005227:Fatty Acids / Q000378:metabolism*; D007700:Kinetics; D016296:Mutagenesis; D019814:Myristic Acid / Q000378:metabolism; D010084:Oxidation-Reduction; D040141:Staphylococcaceae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D000475:Alkenes; D001426:Bacterial Proteins; D005227:Fatty Acids; D019814:Myristic Acid; D003577:Cytochrome P-450 Enzyme System; D002262:Carboxy-Lyases