title: Structure, function and inhibition of ent-kaurene synthase from Bradyrhizobium japonicum.
abstract: We report the first X-ray crystal structure of ent-kaur-16-ene synthase from Bradyrhizobium japonicum, together with the results of a site-directed mutagenesis investigation into catalytic activity. The structure is very similar to that of the α domains of modern plant terpene cyclases, a result that is of interest since it has been proposed that many plant terpene cyclases may have arisen from bacterial diterpene cyclases. The ent-copalyl diphosphate substrate binds to a hydrophobic pocket near a cluster of Asp and Arg residues that are essential for catalysis, with the carbocations formed on ionization being protected by Leu, Tyr and Phe residues. A bisphosphonate inhibitor binds to the same site. In the kaurene synthase from the moss Physcomitrella patens, 16-α-hydroxy-ent-kaurane as well as kaurene are produced since Leu and Tyr in the P. patens kaurene synthase active site are replaced by smaller residues enabling carbocation quenching by water. Overall, the results represent the first structure determination of a bacterial diterpene cyclase, providing insights into catalytic activity, as well as structural comparisons with diverse terpene synthases and cyclases which clearly separate the terpene cyclases from other terpene synthases having highly α-helical structures.
authors.affiliation: 1] Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China [2].
authors.forename: Wenting
authors.initials: W
authors.lastname: Liu
authors.affiliation: 1] Department of Chemistry, University of Illinois, Urbana, IL 61801, USA [2].
authors.forename: Xinxin
authors.initials: X
authors.lastname: Feng
authors.affiliation: 1] Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China [2].
authors.forename: Yingying
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Chun-Hsiang
authors.initials: CH
authors.lastname: Huang
authors.affiliation: Department of Applied Biological Chemistry, Niigata University, Niigata 950-2181, Japan.
authors.forename: Chiaki
authors.initials: C
authors.lastname: Nakano
authors.affiliation: Department of Applied Biological Chemistry, Niigata University, Niigata 950-2181, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Hoshino
authors.affiliation: Department of Chemistry, University of Illinois, Urbana, IL 61801, USA.
authors.forename: Shannon
authors.initials: S
authors.lastname: Bogue
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Tzu-Ping
authors.initials: TP
authors.lastname: Ko
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Chun-Chi
authors.initials: CC
authors.lastname: Chen
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Yunfeng
authors.initials: Y
authors.lastname: Cui
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Li
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Iren
authors.initials: I
authors.lastname: Wang
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Shang-Te Danny
authors.initials: ST
authors.lastname: Hsu
authors.affiliation: Department of Chemistry, University of Illinois, Urbana, IL 61801, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Oldfield
authors.affiliation: Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
authors.forename: Rey-Ting
authors.initials: RT
authors.lastname: Guo
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020369:Bradyrhizobium / Q000201:enzymology*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D045786:Diterpenes, Kaurane / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D045786:Diterpenes, Kaurane; D004791:Enzyme Inhibitors; C002627:kaurene; D019883:Alkyl and Aryl Transferases; C105712:ent-kaurene synthetase B; C108877:terpene synthase