title: Structure and Function of a C-C Bond Cleaving Oxygenase in Atypical Angucycline Biosynthesis.
abstract: C-C bond ring cleaving oxygenases represent a unique family of enzymes involved in the B ring cleavage reaction only observed in atypical angucycline biosynthesis. B ring cleavage is the key reaction leading to dramatic divergence in the final structures of atypical angucyclines. Here, we present the crystal structure of AlpJ, the first structure of this family of enzymes. AlpJ has been verified as the enzyme catalyzing C-C bond cleavage in kinamycin biosynthesis. The crystal structure of the AlpJ monomer resembles the dimeric structure of ferredoxin-like proteins. The N- and C-terminal halves of AlpJ are homologous, and both contain a putative hydrophobic substrate binding pocket in the "closed" and "open" conformations, respectively. Structural comparison of AlpJ with ActVA-Orf6 and protein-ligand docking analysis suggest that the residues including Asn60, Trp64, and Trp181 are possibly involved in substrate recognition. Site-directed mutagenesis results supported our hypothesis, as mutation of these residues led to nearly a complete loss of the activity of AlpJ. Structural analysis also revealed that AlpJ possesses an intramolecular domain-domain interface, where the residues His50 and Tyr178 form a hydrogen bond that probably stabilizes the three-dimensional structure of AlpJ. Site-directed mutagenesis showed that the two residues, His50 and Tyr178, were vital for the activity of AlpJ. Our findings shed light on the structure and catalytic mechanism of the AlpJ family of oxygenases, which presumably involves two active sites that might function in a cooperative manner.
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences , Beijing 100101, People's Republic of China.
authors.forename: Guohui
authors.identifier: 0000-0002-8839-3886
authors.initials: G
authors.lastname: Pan
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences , Beijing 100101, People's Republic of China.|University of Chinese Academy of Sciences , Beijing 100049, People's Republic of China.
authors.forename: Xiaoqin
authors.initials: X
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences , Beijing 100101, People's Republic of China.
authors.forename: Keqiang
authors.identifier: 0000-0003-2420-1889
authors.initials: K
authors.lastname: Fan
authors.affiliation: iHuman Institute, ShanghaiTech University , Shanghai 201210, People's Republic of China.
authors.forename: Junlin
authors.initials: J
authors.lastname: Liu
authors.affiliation: iHuman Institute, ShanghaiTech University , Shanghai 201210, People's Republic of China.
authors.forename: Bing
authors.initials: B
authors.lastname: Meng
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences , Beijing 100101, People's Republic of China.|University of Chinese Academy of Sciences , Beijing 100049, People's Republic of China.
authors.forename: Jinmin
authors.initials: J
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences , Beijing 100101, People's Republic of China.
authors.forename: Bin
authors.initials: B
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences , Beijing 100101, People's Republic of China.|University of Chinese Academy of Sciences , Beijing 100049, People's Republic of China.
authors.forename: Chaobo
authors.initials: C
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences , Beijing 100101, People's Republic of China.
authors.forename: Hui
authors.initials: H
authors.lastname: Han
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences , Beijing 100101, People's Republic of China.
authors.forename: Guomin
authors.initials: G
authors.lastname: Ai
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences , Beijing 100101, People's Republic of China.
authors.forename: Yihua
authors.initials: Y
authors.lastname: Chen
authors.affiliation: iHuman Institute, ShanghaiTech University , Shanghai 201210, People's Republic of China.
authors.forename: Dong
authors.initials: D
authors.lastname: Wu
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences , Beijing 100101, People's Republic of China.|Institute of Molecular and Clinical Medicine, Kunming Medical University , Kunming 650500, China.
authors.forename: Zhi-Jie
authors.initials: ZJ
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences , Beijing 100101, People's Republic of China.
authors.forename: Keqian
authors.identifier: 0000-0001-5203-7938
authors.initials: K
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D008958:Models, Molecular; D010105:Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D061065:Polyketides / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D000072417:Protein Domains; D016415:Sequence Alignment; D013302:Streptomyces / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D061065:Polyketides; D010105:Oxygenases