Two Triterpene Synthases from Imperata cylindrica Catalyzing the Formation of a Pair of Diastereoisomers through Boat or Chair Cyclization.

Imperata cylindrica is known to produce a pair of triterpenes, isoarborinol and fernenol, that exhibit identical planar structures but possess opposite stereochemistry at six of the nine chiral centers. These differences arise from a boat or a chair cyclization of the B-ring of the substrate. Herein, we report the characterization of three OSC genes from I. cylindrica. IcOSC1 and IcOSC5 were identified as isoarborinol and fernenol synthases, respectively, while IcOSC3 was characterized as a multifunctional enzyme that produces glutinol and friedelin as its major products. Mutational studies of isoarborinol and fernenol synthases revealed that the residues surrounding the DCTAE motif partially affected the conformation of the B-ring during cyclization. Additionally, the IcOSC1-W255H mutant produced the rare triterpene boehmerol. The introduced histidine residue presumably abstracted a proton from the intermediary carbocation at C18 during the 1,2-rearrangement. Expression analysis indicated that all OSC genes were highly expressed in stems.

NADH peroxidase plays a crucial role in consuming H2O2 in Lactobacillus casei IGM394.

The facultative anaerobic bacterium Lactobacillus casei IGM394 is used as a host for drug delivery systems, and it exhibits the same growth rate under aerobic and anaerobic conditions. L. casei strains carry several genes that facilitate oxygen and reactive oxygen species (ROS) tolerance in their genomes, but their complete functions have not been uncovered. To clarify the oxygen and ROS tolerance mechanisms of L. casei IGM394, we constructed 23 deficient mutants targeting genes that confer oxidative stress resistance. Significantly decreased growth and high H2O2 accumulation were observed in the NADH peroxidase gene-mutated strain (Δnpr) compared with the findings in the wild type. The H2O2 degradation capacity of Δnpr revealed that NADH peroxidase is a major H2O2-degrading enzyme in L. casei IGM394. Interestingly, ΔohrR, a mutant deficient in the organic hydroperoxide (OhrA) repressor, exhibited higher H2O2 resistance than the wild-type strain. Increased Npr expression and H2O2 degradation ability were observed in ΔohrR, further supporting the importance of OhrA to ROS tolerance mechanisms. The other mutants did not exhibit altered growth rates, although some mutants had higher growth in the presence of oxygen. From these results, it is presumed that L. casei IGM394 has multiple oxygen tolerance mechanisms and that the loss of a single gene does not alter the growth rate because of the presence of complementary mechanisms. Contrarily, the H2O2 tolerance mechanism is solely dependent on NADH peroxidase in L. casei IGM394.

Control of the 1,2-rearrangement process by oxidosqualene cyclases during triterpene biosynthesis.

Oxidosqualene cyclases (OSCs) catalyze the cyclization of an acyclic substrate into various polycyclic triterpenes through a series of cation-π cyclization and 1,2-rearrangement processes. The mechanisms by which OSCs control the fate of intermediate carbocation to generate each specific triterpene product have not yet been determined. The formation of ubiquitous sterol precursors in plants, cycloartenol and Cucurbitaceae-specific cucurbitadienol, only differs by the extent of the 1,2-rearrangement of methyl and hydride. In the present study, we identified critical residues in cycloartenol synthase and cucurbitadienol synthase that were primarily responsible for switching product specificities between the two compounds. The mutation of tyrosine 118 to leucine in cycloartenol synthase resulted in the production of cucurbitadienol as a major product, while the mutation of the corresponding residue leucine 125 to tyrosine in cucurbitadienol synthase resulted in the production of parkeol. Our discovery of this "switch" residue will open up future possibilities for the rational engineering of OSCs to produce the desired triterpenes.