Product Rearrangement from Altering a Single Residue in the Rice syn-Copalyl Diphosphate Synthase

Abstract

Through site-directed mutagenesis targeted at identification of the catalytic base in the rice (Oryza sativa) syn-copalyl diphosphate synthase OsCPS4, changes to a single residue (H501) were found to induce rearrangement rather than immediate deprotonation of the initially formed bicycle, leading to production of the novel compound syn-halimadienyl diphosphate. These mutational results are combined with quantum chemical calculations to provide insight into the underlying reaction mechanism.

Substitution of Two Active‐Site Residues Alters C9‐Hydroxylation in a Class II Diterpene Synthase

“…An additional C4→C5 methyl shift prior to deprotonation will generate the TPP scaffold. However, this latter methyl migration has previously been shown to be energetically more demanding, consistent with the minor occurrence of this reaction observed here . The biosynthesis of 4 is similar in nature to that of a recently described rice CPS4 mutant OsCPS4:H501F/D (corresponding to MvCPS1 F505), which forms syn ‐halima‐5(6),13‐dienyl diphosphate by deprotonation at C6 rather than C5, thereby arriving at the distinct C5=C6 double‐bond isomer .…”

“…However, this latter methyl migration has previously been shown to be energetically more demanding, consistent with the minor occurrence of this reaction observed here . The biosynthesis of 4 is similar in nature to that of a recently described rice CPS4 mutant OsCPS4:H501F/D (corresponding to MvCPS1 F505), which forms syn ‐halima‐5(6),13‐dienyl diphosphate by deprotonation at C6 rather than C5, thereby arriving at the distinct C5=C6 double‐bond isomer . To the best of our knowledge, no enzyme catalysing the formation of 4 has been reported, yet 4 is a minor by‐product of the MvCPS1 wild‐type enzyme and had not been identified prior to this work.…”

A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases

“…Here, we have shown that a single-point mutation that causes an amino acid substitution close to the active site dramatically alters product specificity in both SAD1 and LUP1, so uncovering hidden functional diversity in these triterpene synthases. It is conceivable that nature explores alternate modes of cyclization through such single mutational steps, as has been suggested for diterpene synthases (36)(37)(38)(39)(40)(41)(42)(43). A dedicated cyclase that makes epDM as its major product has not been reported before our work to our knowledge.…”

Diterpene Synthase‐Catalyzed Biosynthesis of Distinct Clerodane Stereoisomers

“…Specifically, the energy profiles for the reactions derived from trans ‐decalin labda‐13 E ‐en‐8‐yl + diphosphate intermediates and from cis ‐decalin labda‐13 Z ‐en‐8‐yl + diphosphate intermediates were calculated to predict inherent energetic barriers (Figure ) . Results from these calculations (see Figure S6 for details) indicate that the final 1,2‐methyl shift has the highest energetic barrier in all cases; this is consistent with previous studies on the GGPPderived ent ‐ and syn ‐ labda‐13 E ‐en‐8‐yl + intermediate reactions leading to CLPP . Differences in transition‐state energies for 1,2‐shifts of the C‐18 and C‐19 methyl groups were small (ca.…”