Unlike EC 4.2.3.29, ent-sandaracopimaradiene synthase, which can produce both ent-sandaracopimaradiene and ent-pimara-8(14),15-diene, this diterpene cyclase produces only ent-pimara-8(14),15-diene. ent-Pimara-8(14),15-diene is not a precursor in the biosynthesis of either gibberellins or phytoalexins.
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The enzyme appears in viruses and cellular organisms
Unlike EC 4.2.3.29, ent-sandaracopimaradiene synthase, which can produce both ent-sandaracopimaradiene and ent-pimara-8(14),15-diene, this diterpene cyclase produces only ent-pimara-8(14),15-diene. ent-Pimara-8(14),15-diene is not a precursor in the biosynthesis of either gibberellins or phytoalexins.
GC-MS reaction product analysis. The distinct function of two alleles of a KSL from rice, OsKSL5i and OsKSLj, identifies a single residue switch with a profound effect on not only OsKSL5 product outcome but also that of land plant KSs more broadly, specifically, replacement of a key isoleucine with threonine, which interrupts formation of the tetracyclic ent-isokaurene at the tricyclic stage, leading to production of ent-pimaradiene instead
the OsKSL5 mutant I664A produces almost exclusively ent-pimara-8(14),15-diene, it also produces low amounts of ent-copalol, the dephosphorylated derivative of ent-copalyl diphosphate
substitution of Ile for the Thr found at this position in OsKSL5j does not lead to complete specificity for production of ent-isokaur-15-ene, as the resulting OsKSL5j:T664I also produces small amounts of ent-kaur-16-ene, along with more substantial amounts of ent-atiserene, resulting from alternative rearrangement of the ent-beyeran-12-yl+ intermediate formed by the second cyclization step
conserved residues at positions 661 (secondary) and 664 (primary) are responsible for substrate specificity. Two functionally distinct alleles of Oryza sativa OsKSL5, which react with ent-copalyl diphosphate (ent-copalyl diphosphate), with that from subspecies japonica (OsKSL5j) producing ent-pimara-8(14),15-diene and that from subspecies indica (OsKSL5i) producing ent-(iso)kaur-15-ene instead
in the plant kingdom, where the labdane-related diterpenoids (LRDs) are predominantly found, the class I and II diterpene cyclases seem to have most directly evolved via gene duplication and neofunctionalization of the ent-kaurene synthases (KSs) required in all vascular plants for gibberellin hormone biosynthesis. Accordingly, these enzymes are often termed KS-like (KSL) and form a distinct subfamily within the plant terpene synthase family. Like class I terpene synthases more generally, the KS(L)s carry out catalysis in a highly conserved alpha-helical bundle domain that contains two signature motifs. These DDxxD and NDxx(S/T)xxxE sequences are involved in ligation of a trinuclear Mg2+ cluster to promote substrate binding and the subsequent initiating ionization of the allylic diphosphate ester bond. Ile664 is conserved across all the known plant KSs, and substitution of Thr is found to similarly alter product outcome with the KS from not only rice (OsKS) but also the eudicot Arabidopsis thaliana (AtKS), leading to predominant production of ent-pimara-8(14),15-diene rather than ent-kaur-16-ene. OsKSL5 belongs to an early diverging KSL lineage within monocots and is relatively distantly related to any KSs
enzymes KSLs belong to the type A/class I terpene synthase family. The KSL5 members are clearly divided into two clades, representing japonica and indica groups. The KS responsible for gibberellin biosynthesis in a putative common ancestor, and OsKS1, which produces the tetracyclic ent-kaurene, has Ile as the corresponding key amino acid. Thus, it is suggested that substitution from Ile664 to Thr664 in KSL5 occurred before the onset of cultivation in the putative japonica ancestor. The OsKSL5 sequence is similar to the OsKSL6 sequence (89% identity), and it is therefore likely that the two KSL genes were generated through relatively recent duplication. The substitution in KSL5 probably occurred after duplication of the ancestral gene
recombinant OrKSL5 converts ent-CDP to ent-pimara-8(14),15-diene (EC 4.2.3.30), whereas OrKSL5ind, OrKSL6 and OrKSL6ind all convert ent-CDP to ent-isokaurene (EC 4.2.3.103). The 664th amino acids of OrKSL5 and OrKSL5ind are Thr and Ile, respectively, as is the case for Oryza sativa OsKSL5 and OsKSL5ind
the KSL5 members are clearly divided into two clades, representing japonica and indica groups, the 664th amino acids of OsKSL5j and OsKSL5i are Thr and Ile leading to different activity and production of ent-isokeurene synthase (EC 4.2.3.103) or ent-pimara-8(14),15-diene (EC 4.2.3.30)
site-directed mutagenesis, the addition of L661Vto mutation T664I leads to much more specific production of ent-isokaurene, the product outcome mediated by this OsKSL5j:L661V/T664I double residue switch mutant is essentially identical to that mediated by OsKSL5i
of the three differences in active site residues between the orthologous OsKSL5j and OsKSL5i, switching the Ile at position 664 in OsKSL5i to the Thr found in OsKSL5j is sufficient to short circuit the production of ent-isokaurene, as OsKSL5i:I664T specifically produces ent-pimara-8(14),15-diene instead. But the reciprocal residue switch leads to production of a mixture of tetracycles ent-isokaurene, ent-kaurene, and ent-atiserene (i.e. by OsKSL5j:T664I), rather than the almost exclusive production of ent-isokaurene observed with OsKSL5i
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene AN1594, the AN1594 overexpression strain A772 oe:AN1594 is created by placing AN1594 under the control of the constitutively active gpdA promoter in Aspergillus nidulans strain A772, quantitative RT-PCR enzyme expression analysis
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
wild-type Aspergillus nidulans strain overexpressing the cluster-specific transcription factor, pbcR, shows upregulation of the cluster genes leading to ent-pimara-8(14),15-diene production in Aspergillus nidulans and 11.7fold increased expression of gene ANIA_01594
wild-type Aspergillus nidulans strain overexpressing the cluster-specific transcription factor, pbcR, shows upregulation of the cluster genes leading to ent-pimara-8(14),15-diene production in Aspergillus nidulans and 11.7fold increased expression of gene ANIA_01594
wild-type Aspergillus nidulans strain overexpressing the cluster-specific transcription factor, pbcR, shows upregulation of the cluster genes leading to ent-pimara-8(14),15-diene production in Aspergillus nidulans and 11.7fold increased expression of gene ANIA_01594
wild-type Aspergillus nidulans strain overexpressing the cluster-specific transcription factor, pbcR, shows upregulation of the cluster genes leading to ent-pimara-8(14),15-diene production in Aspergillus nidulans and 11.7fold increased expression of gene ANIA_01594
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4.2.3.30 ent-pimara-8(14),15-diene synthase
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