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Literature summary extracted from

  • Jia, M.; Zhou, K.; Tufts, S.; Schulte, S.; Peters, R.J.
    A pair of residues that interactively affect diterpene synthase product outcome (2017), ACS Chem. Biol., 12, 862-867 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
4.2.3.30 sequence comparisons and phylogenetic analysis and tree Oryza sativa Japonica Group
4.2.3.103 gene KSL5i, sequence comparisons and phylogenetic analysis and tree, recombinant expression of His-tagged wild-type and mutant enzymes without the N-terminal transit peptide sequence in Escherichia coli Oryza sativa Indica Group
4.2.3.103 gene KSL6, sequence comparisons and phylogenetic analysis and tree, recombinant expression of His-tagged wild-type and mutant enzymes without the N-terminal transit peptide sequence in Escherichia coli Oryza sativa Japonica Group

Protein Variants

EC Number Protein Variants Comment Organism
4.2.3.30 additional information 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 Oryza sativa Japonica Group
4.2.3.30 T664I site-directed mutagenesis, the OsKSL5j:T664I mutant produces only ent-pimara-8(14),15-diene Oryza sativa Japonica Group
4.2.3.30 T664I/L661V 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 Oryza sativa Japonica Group
4.2.3.30 T664I/V718I site-directed mutagenesis, the addition of V718I to mutation T664I does not further alter product outcome Oryza sativa Japonica Group
4.2.3.103 I664T site-directed mutagenesis, the OsKSL5i:I664T mutant produces only ent-pimara-8(14),15-diene Oryza sativa Indica Group
4.2.3.103 additional information 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 Oryza sativa Indica Group
4.2.3.103 V661L site-directed mutagenesis, substitution of Leu for the Val found at this secondary position only slightly decreases catalytic fidelity, the mutant still predominantly produces ent-isokaurene, with only slight increases in the relative amounts of ent-kaur-16-ene and ent-atiserene Oryza sativa Japonica Group
4.2.3.103 V661L site-directed mutagenesis, the mutant single residue switch, OsKSL5i:V661L, leads to reduced catalytic specificity and production of a mixture of ent-isokaurene, ent-keurene, and ent-atiserene, closely resembling that mediated by the OsKSL5j:T664I mutant (EC 4.2.3.30), although it does not affect the specific production of ent-pimara-8(14),15-diene Oryza sativa Indica Group

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
4.2.3.30 chloroplast the enzyme contains an N-terminal transit peptide sequence Oryza sativa Japonica Group 9507
-
4.2.3.103 chloroplast the enzyme contains a N-terminal transit peptide sequence Oryza sativa Japonica Group 9507
-
4.2.3.103 chloroplast the enzyme contains a N-terminal transit peptide sequence Oryza sativa Indica Group 9507
-

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
4.2.3.30 Mg2+ required Oryza sativa Japonica Group
4.2.3.103 Mg2+ required Oryza sativa Japonica Group
4.2.3.103 Mg2+ required Oryza sativa Indica Group

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
4.2.3.30 ent-copalyl diphosphate Oryza sativa Japonica Group
-
ent-pimara-8(14),15-diene + diphosphate
-
?
4.2.3.103 ent-copalyl diphosphate Oryza sativa Japonica Group
-
ent-isokaurene + diphosphate
-
?
4.2.3.103 ent-copalyl diphosphate Oryza sativa Indica Group
-
ent-isokaur-15-ene + diphosphate
-
?

Organism

EC Number Organism UniProt Comment Textmining
4.2.3.30 Oryza sativa Japonica Group Q6Z5J6
-
-
4.2.3.103 Oryza sativa Indica Group A4KAG7
-
-
4.2.3.103 Oryza sativa Japonica Group A4KAG8
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
4.2.3.103 recombinant His-tagged wild-type and mutant enzymes without the N-terminal transit peptide sequence from Escherichia coli by nickel affinity chromatography Oryza sativa Japonica Group
4.2.3.103 recombinant His-tagged wild-type and mutant enzymes without the N-terminal transit peptide sequence from Escherichia coli by nickel affinity chromatography Oryza sativa Indica Group

Source Tissue

EC Number Source Tissue Comment Organism Textmining
4.2.3.30 leaf
-
Oryza sativa Japonica Group
-
4.2.3.103 leaf
-
Oryza sativa Japonica Group
-
4.2.3.103 leaf
-
Oryza sativa Indica Group
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.2.3.30 ent-copalyl diphosphate
-
Oryza sativa Japonica Group ent-pimara-8(14),15-diene + diphosphate
-
?
4.2.3.30 additional information 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 Oryza sativa Japonica Group ?
-
?
4.2.3.103 ent-copalyl diphosphate
-
Oryza sativa Japonica Group ent-isokaurene + diphosphate
-
?
4.2.3.103 ent-copalyl diphosphate
-
Oryza sativa Indica Group ent-isokaur-15-ene + diphosphate
-
?
4.2.3.103 additional information 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 Oryza sativa Indica Group ?
-
?

Synonyms

EC Number Synonyms Comment Organism
4.2.3.30 kaurene synthase-like enzyme 5
-
Oryza sativa Japonica Group
4.2.3.30 KS-like 5j
-
Oryza sativa Japonica Group
4.2.3.30 OsKSL5j
-
Oryza sativa Japonica Group
4.2.3.103 kaurene synthase-like enzyme 5
-
Oryza sativa Indica Group
4.2.3.103 kaurene synthase-like enzyme 6
-
Oryza sativa Japonica Group
4.2.3.103 KS-like 5i
-
Oryza sativa Indica Group
4.2.3.103 KS-like 6
-
Oryza sativa Japonica Group
4.2.3.103 OsKSL5i
-
Oryza sativa Indica Group
4.2.3.103 OsKSL6
-
Oryza sativa Japonica Group

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
4.2.3.103 37
-
assay at Oryza sativa Japonica Group
4.2.3.103 37
-
assay at Oryza sativa Indica Group

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
4.2.3.103 7
-
assay at Oryza sativa Japonica Group
4.2.3.103 7
-
assay at Oryza sativa Indica Group

General Information

EC Number General Information Comment Organism
4.2.3.30 evolution 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 Oryza sativa Japonica Group
4.2.3.30 malfunction 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 Oryza sativa Japonica Group
4.2.3.30 additional information 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 Oryza sativa Japonica Group
4.2.3.103 evolution 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 Oryza sativa Japonica Group
4.2.3.103 evolution 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. OsKSL5 belongs to an early diverging KSL lineage within monocots and is relatively distantly related to any KSs Oryza sativa Indica Group
4.2.3.103 additional information 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, EC 4.2.3.30) producing ent-pimara-8(14),15-diene and that from subspecies indica (OsKSL5i, EC 4.2.3.103) producing ent-(iso)kaur-15-ene instead Oryza sativa Indica Group