title: Crystal Structure of StnA for the Biosynthesis of Antitumor Drug Streptonigrin Reveals a Unique Substrate Binding Mode.
abstract: Streptonigrin methylesterase A (StnA) is one of the tailoring enzymes that modify the aminoquinone skeleton in the biosynthesis pathway of Streptomyces species. Although StnA has no significant sequence homology with the reported α/β-fold hydrolases, it shows typical hydrolytic activity in vivo and in vitro. In order to reveal its functional characteristics, the crystal structures of the selenomethionine substituted StnA (SeMet-StnA) and the complex (S185A mutant) with its substrate were resolved to the resolution of 2.71 Å and 2.90 Å, respectively. The overall structure of StnA can be described as an α-helix cap domain on top of a common α/β hydrolase domain. The substrate methyl ester of 10'-demethoxystreptonigrin binds in a hydrophobic pocket that mainly consists of cap domain residues and is close to the catalytic triad Ser185-His349-Asp308. The transition state is stabilized by an oxyanion hole formed by the backbone amides of Ala102 and Leu186. The substrate binding appears to be dominated by interactions with several specific hydrophobic contacts and hydrogen bonds in the cap domain. The molecular dynamics simulation and site-directed mutagenesis confirmed the important roles of the key interacting residues in the cap domain. Structural alignment and phylogenetic tree analysis indicate that StnA represents a new subfamily of lipolytic enzymes with the specific binding pocket located at the cap domain instead of the interface between the two domains.
authors.affiliation: State Key Laboratory of Microbial metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, China.
authors.forename: Tianle
authors.initials: T
authors.lastname: Qian
authors.affiliation: State Key Laboratory of Microbial metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Wo
authors.affiliation: State Key Laboratory of Microbial metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, China.|Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai, 200025, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Microbial metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, China.|Key laboratory of Pesticide and Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, 152 Luoyu Road, Wuhan 430079, China.
authors.forename: Quanwei
authors.initials: Q
authors.lastname: Song
authors.affiliation: Key laboratory of Pesticide and Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, 152 Luoyu Road, Wuhan 430079, China.
authors.forename: Guoqiang
authors.initials: G
authors.lastname: Feng
authors.affiliation: Departments of Molecular Biology and Biochemistry, Chemical Engineering and Materials Science, and Biomedical Engineering, University of California, Irvine, California 92697-3900, USA.
authors.forename: Ray
authors.initials: R
authors.lastname: Luo
authors.affiliation: State Key Laboratory of Microbial metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, China.
authors.forename: Shuangjin
authors.initials: S
authors.lastname: Lin
authors.affiliation: State Key Laboratory of Microbial metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, China.
authors.forename: Geng
authors.initials: G
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Microbial metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, China.|Shanghai Center for Bioinformation Technology, 1278 Keyuan Road, Shanghai, 200235, China.
authors.forename: Hai-Feng
authors.initials: HF
authors.lastname: Chen
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000737:chemistry* / Q000493:pharmacokinetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000648:ultrastructure; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000648:ultrastructure; D020134:Catalytic Domain; D004926:Escherichia coli; D008968:Molecular Conformation; D056004:Molecular Dynamics Simulation; D020539:Sequence Analysis, Protein; D013308:Streptonigrin / Q000096:biosynthesis* / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D000903:Antibiotics, Antineoplastic; D001426:Bacterial Proteins; D013308:Streptonigrin; D002265:Carboxylic Ester Hydrolases; C000628803:StnA protein, Streptomyces sp