title: Enzymatic [4+2] Cycloadditions in the Biosynthesis of Spirotetramates and Spirotetronates.
abstract: The Diels-Alder reaction is a quintessential type of [4+2] cycloaddition that remains one of the most intriguing transformations in synthetic chemistry. This reaction has long been envisaged to participate in the biosynthesis of a number of cyclohexene-containing natural products, although the question of whether a bona fide Diels-Alderase exists remains unsolved. In nature, there are remarkably few enzymes known to have the activity of [4+2] cycloaddition. These enzymes are phylogenetically distinct and are often classified according to the specific chemical structures. The variation of protein ancestors and in many cases the instability/complexity of the substrates and products pose a significant challenge in identification of the [4+2] cycloaddition catalysts using general homology-based mining approaches. We here provide the detailed description of the multiple comparison-based strategy and methods for the characterization of two distinct types of dedicated [4+2] cyclases (eg, PyrE3 and PyrI4) in the biosynthesis of spirotetramates and spirotetronates, where they act in tandem for coordinated cross-bridging of a linear polyene intermediate into a enantiomerically pure pentacyclic core. The search of new protein scaffolds with the [4+2] cycloaddition activity could enrich the pool of the candidates for mechanistic examination of a true enzymatic Diels-Alder reaction. The protocols presented in this study would also be applicable to the study of other functionally similar but phylogenetically different proteins, eg, the spiroketal cyclases.
authors.affiliation: State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, PR China.
authors.forename: B
authors.initials: B
authors.lastname: Pang
authors.affiliation: State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, PR China.
authors.forename: G
authors.initials: G
authors.lastname: Zhong
authors.affiliation: State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, PR China.
authors.forename: Z
authors.initials: Z
authors.lastname: Tang
authors.affiliation: State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, PR China; Huzhou Center of Bio-Synthetic Innovation, Huzhou, PR China. Electronic address: wliu@mail.sioc.ac.cn.
authors.forename: W
authors.initials: W
authors.lastname: Liu
mesh_terms: D000617:Aminoglycosides / Q000235:genetics / Q000378:metabolism*; D053898:Biosynthetic Pathways; D003500:Cyclization; D061565:Cycloaddition Reaction; D005663:Furans / Q000378:metabolism; D008025:Ligases / Q000235:genetics / Q000378:metabolism; D008190:Lyases / Q000235:genetics / Q000378:metabolism; D018942:Macrolides / Q000378:metabolism*; D005810:Multigene Family; D013141:Spiro Compounds / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000617:Aminoglycosides; D005663:Furans; D018942:Macrolides; D013141:Spiro Compounds; C091121:pyrroindomycin A; C520774:spiroketal; D008190:Lyases; D008025:Ligases; C000451:chlorothricin
keywords: Dialkyldecalin synthase; Diels–Alder reaction; Spiroconjugate synthase; Spiroketal cyclase; Spirotetramate; Spirotetronate; [4+2] Cycloaddition