geranylgeranyl-pyrophosphate and copalyl-diphosphate

The traditional Chinese medicinal plant, Isodon L., is remarkably rich in pharmacologically active ent-kaurane diterpenoids of diverse carbon skeletons. In an effort to create a resource for gene discovery and elucidate the biosynthesis of Isodonent-kaurane diterpenoids, three cDNAs (named IeCPS1, IeCPS2 and IeCPS2a) were isolated putatively encoding copalyl diphosphate synthases from Isodoneriocalyx leaves. Recombinant proteins of IeCPS1 and IeCPS2 were expressed, respectively, in Escherichia coli, and were shown to specifically convert geranylgeranyl diphosphate to copalyl diphosphate as demonstrated by GC-MS analyses. Based on tissue-specific expression and metabolic localization studies, the IeCPS2 transcripts were detected in young and mature leaves where the dominant ent-kaurane diterpenoid maoecrystal B accumulates, whereas no detectable expression of IeCPS2 was observed in germinating seeds where the gibberellin biosynthetic pathway is usually active. In addition, no evidence for maoecrystal B was found in germinating seeds. On the other hand, IeCPS1 transcripts significantly accumulated in germinating seeds as well as in leaves. The biochemical and molecular genetic evidence thus indicated that IeCPS2 is a copalyl diphosphate synthase potentially involved in the biosynthesis of Isodon diterpenoids in leaves, while IeCPS1 is more probably relevant to gibberellin formation and may, in addition, participate in Isodonent-kaurane diterpenoid production.

Topics: Alkyl and Aryl Transferases; Amino Acid Sequence; Cloning, Molecular; Diterpenes, Kaurane; DNA, Complementary; Enzyme Activation; Escherichia coli; Gas Chromatography-Mass Spectrometry; Germination; Gibberellins; Isodon; Medicine, Chinese Traditional; Molecular Sequence Data; Organophosphates; Phylogeny; Plant Leaves; Plant Proteins; Polyisoprenyl Phosphates; Recombinant Proteins; Seeds; Substrate Specificity

Class II diterpene cyclases catalyze bicyclization of geranylgeranyl diphosphate. While this reaction typically is terminated via methyl deprotonation to yield copalyl diphosphate, in rare cases hydroxylated bicycles are produced instead. Abietadiene synthase is a bifunctional diterpene cyclase that usually produces a copalyl diphosphate intermediate. Here it is shown that substitution of aspartate for a conserved histidine in the class II active site of abietadiene synthase leads to selective production of 8α-hydroxy-CPP instead, demonstrating striking plasticity.

Topics: Abies; Aspartic Acid; Catalysis; Cyclization; Diterpenes; Histidine; Hydroxylation; Isomerases; Molecular Structure; Organophosphates; Polyisoprenyl Phosphates

Abietadiene synthase from grand fir catalyzes two sequential, mechanistically distinct cyclizations, of geranylgeranyl diphosphate and of copalyl diphosphate, in the formation of a mixture of abietadiene isomers as the committed step of diterpenoid resin acid biosynthesis. Each reaction is independently conducted at a separate active site residing in what were considered to be structurally distinct domains typical of terpene cyclases. Despite the presence of an unusual 250-residue N-terminal insertional element, a tandem pair of charged residues distal to the insertion was shown to form a functional part of the C-terminal active site. Because abietadiene synthase resembles the ancestral plant terpene cyclase, this observation suggests an early evolutionary origin of catalytically important positively charged residues at the N-terminus of enzymes of this general class. A series of N- and C-terminal truncations of this enzyme were constructed and characterized, both alone and as mixtures of adjacent polypeptide pairs, to assess the proposed domain architecture, the function of the insertional element, and the role of presumptive interdomain contacts. These studies indicated a requirement for the insertional element in functional folding and allowed definition of the minimum primary structure of N- and C-terminal active site peptides. Most importantly, the results showed that, although the two active sites of abietadiene synthase are catalytically independent, substantial contact between the two regions is essential for the functional competence of this enzyme. Thus, the two cyclization sites of abietadiene synthase cannot be dissected into catalytically distinct domains, and, therefore, abietadiene synthase is unlikely to have arisen by fusion of two previously independent genes.

Topics: Abies; Binding Sites; Catalysis; Diterpenes; DNA Mutational Analysis; Ions; Isomerases; Models, Chemical; Models, Molecular; Multienzyme Complexes; Organophosphates; Peptide Fragments; Plant Proteins; Polyisoprenyl Phosphates; Protein Structure, Secondary; Protein Structure, Tertiary; Protons; Sequence Deletion; Structure-Activity Relationship

Abietadiene synthase catalyzes two sequential, mechanistically distinct cyclization reactions in the formation of a mixture of abietadiene double bond isomers as the committed step in resin acid biosynthesis. Each reaction is carried out at a separate active site residing in a structurally distinct domain, and the reactions are kinetically separable. The first cyclization reaction is initiated by protonation of the terminal double bond of the universal diterpene precursor, geranylgeranyl diphosphate. The pH dependence of the overall reaction is consistent with an acid-base catalytic mechanism, and a divalent metal ion plays a role in this reaction probably by binding the diphosphate moiety to assist in positioning the substrate for catalysis. A putative active site for the protonation-initiated cyclization was defined by modeling abietadiene synthase and locating the DXDD motif previously shown to be involved in this reaction. A number of charged and aromatic residues, which are highly conserved in mechanistically related diterpene cyclases, line the putative active site. Alanine substitutions were made for each of these residues, as were asparagine and glutamate substitutions for the aspartates of the DXDD motif. Kinetic evaluation confirmed the involvement of most of the targeted residues in the reaction, and analysis of mutational effects on the pH-activity profile and affinity for a transition state analogue suggested specific roles for several of these residues in catalyzing the cyclization of geranylgeranyl diphosphate to (+)-copalyl diphosphate. A functional role was also suggested for the cryptic insertional element found in abietadiene synthase and other diterpene synthases that carry out similar protonation-initiated cyclizations.

Topics: Amino Acid Motifs; Amino Acid Sequence; Catalytic Domain; Conserved Sequence; Diterpenes; Hydrogen-Ion Concentration; Isomerases; Models, Molecular; Mutagenesis, Site-Directed; Organophosphates; Polyisoprenyl Phosphates; Protons; Substrate Specificity; Trees

Abietadiene synthase (AS) catalyzes two sequential, mechanistically distinct cyclizations in the conversion of geranylgeranyl diphosphate to a mixture of abietadiene double bond isomers as the initial step of resin acid biosynthesis in grand fir (Abies grandis). The first reaction converts geranylgeranyl diphosphate to the stable bicyclic intermediate (+)-copalyl diphosphate via protonation-initiated cyclization. In the second reaction, diphosphate ester ionization-initiated cyclization generates the tricyclic perhydrophenanthrene-type backbone, and is directly coupled to a 1,2-methyl migration that generates the C13 isopropyl group characteristic of the abietane family of diterpenes. Using the transition-state analogue inhibitor 14,15-dihydro-15-azageranylgeranyl diphosphate, it was demonstrated that each reaction of abietadiene synthase is carried out at a distinct active site. Mutations in two aspartate-rich motifs specifically delete one or the other activity and the location of these motifs suggests that the two active sites reside in separate domains. These mutants effectively complement each other, suggesting that the copalyl diphosphate intermediate diffuses between the two active sites in this monomeric enzyme. Free copalyl diphosphate was detected in steady-state kinetic reactions, thus conclusively demonstrating a free diffusion transfer mechanism. In addition, both mutant enzymes enhance the activity of wild-type abietadiene synthase with geranylgeranyl diphosphate as substrate. The implications of these results for the kinetic mechanism of abietadiene synthase are discussed.

Topics: Amino Acid Motifs; Aspartic Acid; Aza Compounds; Binding Sites; Isomerases; Kinetics; Mutagenesis, Site-Directed; Organophosphates; Polyisoprenyl Phosphates; Stereoisomerism; Trees