Literature summary for 5.4.99.39 extracted from

Hoshino, T.; Nakagawa, K.; Aiba, Y.; Itoh, D.; Nakada, C.; Masukawa, Y.
Euphorbia tirucalli beta-amyrin synthase critical roles of steric sizes at Val483 and Met729 and the CH-Pi interaction between Val483 and Trp534 for catalytic action (2017), ChemBioChem, 18, 2145-2155 .

Search for more literature for 5.4.99.39

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of wild-type and mutant enzymes in Saccharomyces cerevisiae strain GIL77	Euphorbia tirucalli

Protein Variants

Protein Variants	Comment	Organism
M729A	site-directed mutagenesis, the mutant shows a decreased enzymatic activity compared to wild-type and altered roduct distribution ratio	Euphorbia tirucalli
M729F	site-directed mutagenesis, the mutant shows a decreased enzymatic activity compared to wild-type and altered roduct distribution ratio	Euphorbia tirucalli
M729G	site-directed mutagenesis, the mutant shows a decreased enzymatic activity compared to wild-type and altered roduct distribution ratio	Euphorbia tirucalli
M729L	site-directed mutagenesis, the mutant shows an unaltered enzymatic activity compared to wild-type and altered roduct distribution ratio	Euphorbia tirucalli
M729N	site-directed mutagenesis, the mutant shows a decreased enzymatic activity compared to wild-type and altered roduct distribution ratio	Euphorbia tirucalli
M729V	site-directed mutagenesis, the mutant shows a decreased enzymatic activity compared to wild-type and altered roduct distribution ratio	Euphorbia tirucalli
M729W	site-directed mutagenesis, the mutant with the bulky substitution is inactive	Euphorbia tirucalli
V483A	site-directed mutagenesis, the mutant shows altered substrate specificity compared to wild-type, and affords monocyclic camelliol C	Euphorbia tirucalli
V483F	site-directed mutagenesis	Euphorbia tirucalli
V483G	site-directed mutagenesis, the mutant shows altered substrate specificity compared to wild-type, and affords monocyclic camelliol C	Euphorbia tirucalli
V483I	site-directed mutagenesis, the mutant shows unaltered substrate specificity compared to wild-type, and affords beta-amyrin as major product	Euphorbia tirucalli
W534A	site-directed mutagenesis, the mutant shows significantly decreased enzymatic activity compared to wild-type and provides no aberrantly cyclized product	Euphorbia tirucalli
W534F	site-directed mutagenesis, the mutant shows significantly decreased enzymatic activity compared to wild-type and provides no aberrantly cyclized product	Euphorbia tirucalli
W534H	site-directed mutagenesis, the mutant shows significantly decreased enzymatic activity compared to wild-type and provides no aberrantly cyclized product	Euphorbia tirucalli
W534I	site-directed mutagenesis, the mutant shows significantly decreased enzymatic activity compared to wild-type and provides no aberrantly cyclized product	Euphorbia tirucalli
W534M	site-directed mutagenesis, the mutant shows significantly decreased enzymatic activity compared to wild-type and provides no aberrantly cyclized product	Euphorbia tirucalli
W534V	site-directed mutagenesis, the mutant shows significantly decreased enzymatic activity compared to wild-type and provides no aberrantly cyclized product	Euphorbia tirucalli
W534Y	site-directed mutagenesis, the mutant shows significantly decreased enzymatic activity compared to wild-type and provides no aberrantly cyclized product	Euphorbia tirucalli

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
(3S)-2,3-epoxy-2,3-dihydrosqualene	Euphorbia tirucalli	-	beta-amyrin	-	r

Organism

Organism	UniProt	Comment	Textmining
Euphorbia tirucalli	Q401R6	-	-

Reaction

Reaction	Comment	Organism	Reaction ID
(3S)-2,3-epoxy-2,3-dihydrosqualene = beta-amyrin	cyclization pathway of (3S)-2,3-oxidosqualene to generate beta-amyrin triterpene. (3S)-2,3-oxidosqualene is folded into a chair-chair-chair-boat-boat conformation in the enzyme cavity, and the proton released from the DCTA amino acid motif attacks the epoxide ring, leading to sequential ring-forming reactions and the construction of 6,6,6,6,6-fused pentacyclic ring scaffold via several carbocationic intermediates. The oleanyl cation undergoes rearrangement reactions of 1,2-hydride shifts and deprotonation of axial-oriented H-12 to yield beta-amyrin	Euphorbia tirucalli	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
(3S)-2,3-epoxy-2,3-dihydrosqualene	-	Euphorbia tirucalli	beta-amyrin	-	r

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Euphorbia tirucalli

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7	-	assay at	Euphorbia tirucalli

General Information

General Information	Comment	Organism
malfunction	the Gly and Ala variants with a smaller bulk size at position 483, compared to wild-type Val483, predominantly afford monocyclic camelliol C, which suggests that the orientation of the (3S)-2,3-oxidosqualene substrate is not appropriately arranged in the reaction cavity as a result of the decreased bulk size, leading to failure of its normal folding into the chair-chair-chair-boat-boat conformation. The Ile variant, with a somewhat larger bulk, affords beta-amyrin as the dominant product. Various point mutants of Trp534 exhibit significantly decreased enzymatic activities and provide no aberrantly cyclized products, although the aromatic Phe and Tyr residues are incorporated and the steric sizes of the aliphatic residues are altered. Therefore, the Trp534 residue does not stabilize the transient cation through a cation-Pi interaction. Altering the steric bulk at the Met729 position afforded the pentacyclic skeletons	Euphorbia tirucalli
additional information	the Trp534 residue does not stabilize the transient cation through a cation-Pi interaction. the Trp residue, with the largest steric bulk among all natural amino acids, is essential for high enzymatic activity. Robust CH-Pi complexation between the Val483 and Trp534 residues is proposed. Met729 is positioned at the E-ring formation site. Homology modeling and structure-function analysis, overview	Euphorbia tirucalli

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Release 2023.1 (January 2023)