Letter Cite This: Org. Lett. 2018, 20, 1802−1805
pubs.acs.org/OrgLett
Astertarone A Synthase from Chinese Cabbage Does Not Produce the C4-Epimer: Mechanistic Insights Jing Jin,† Megan K. Moore,‡ William K. Wilson,† and Seiichi P. T. Matsuda*,†,‡ †
Department of BioSciences and ‡Department of Chemistry, Rice University, Houston, Texas 77005, United States S Supporting Information *
ABSTRACT: The Brassica rapa oxidosqualene cyclase Bra032185 makes 60% astertarone A (1) and 20 minor triterpenes (0.1−11%). Mechanistic analysis indicates the absence of an enol intermediate to generate the 4S epimer of 1, unless workup involves saponification. Bra032185 and its closest Arabidopsis thaliana homologue diverged markedly in product profiles over a short evolutionary distance, while developing opposite C18 configurations. Active-site residue comparison of Bra032185 with friedelin and shionone synthases suggests convergent evolution to 3-ketotriterpenes.
T
riterpene synthases convert (3S)-2,3-oxidosqualene and squalene into more than 100 triterpene skeletons, which may be further metabolized to over 30 000 triterpenoid natural products of biological and medicinal importance.1 Beyond primary metabolism with cycloartenol synthase, most higher plants contain additional oxidosqualene cyclases (OSCs) to produce the diverse triterpenes of secondary metabolism.2 The major product of OSCs is routinely predicted through bioinformatics but usually without experimental confirmation. Comparing OSCs from related species provides insight into their complex evolutionary history. Since completion of the genome for the model plant Arabidopsis thaliana, several other genomes from the Brassicaceae family have been sequenced, including Brassica rapa.3 Whereas product profiles of all 13 OSCs of A. thaliana have been reported,4 no B. rapa OSC product profile has been experimentally characterized. B. rapa is agriculturally important for its vegetable cultivars (turnip and cabbage) and oilseed crops. Brassica species (in Brassicaceae family Lineage II) are the closest crop relatives to A. thaliana (in Lineage I).5 Here, we describe the product profile of a B. rapa OSC (Bra032185, from Chinese cabbage) as an initial step toward cataloging the triterpenes these crops can produce and understanding the evolution of triterpenoid biosynthesis in Arabidopsis and Brassica species. A BLAST search with A. thaliana OSC sequences against the B. rapa genome provided several putative OSCs. Among these candidates for study, Bra032185 (XM_009142199, bioinformatically annotated as tirucalladienol synthase-like enzyme) was intriguing because of its moderate similarity (79% amino acid sequence identity) to its closest homologue in A. thaliana (AtPEN3). AtPEN3 makes ∼85% tirucalla-7,24-dien-3β-ol4 and is clustered together with Bra032185 in the phylogenetic tree, shown in Figure 1. In contrast, Bra032185 has close Brassica homologues expected to share similar product profiles: © 2018 American Chemical Society
Figure 1. Maximum likelihood tree of Bra032185 with A. thaliana OSCs using the cycloartenol synthase (CAS) from Amborella trichopoda as the outgroup (1000 replicates; bootstrap percentages are given at the nodes). The tree was constructed using amino acid sequences with MEGA version 6.6
BnaA04g13460D from Brassica napus (99% identity) and XP_013633230 from Brassica oleracea (97% identity). The product profile of Bra032185 (Figure 2) was determined by standard heterologous expression in yeast. After GenScript synthesized a version of Bra032185 codon-optimized for Saccharomyces cerevisiae, we subcloned the gene into the yeast expression vector pRS426Gal.7 The resulting plasmid was expressed in S. cerevisiae strains EHY418 and RXY6.9 After workup, the yeast extracts were purified chromatographically and analyzed by NMR and GC-MS, as described in the Supporting Information (SI). Three independent experiments (Table 1) showed astertarone A (1) as the major product of Bra032185, accompanied by 20 Received: January 28, 2018 Published: March 20, 2018 1802
DOI: 10.1021/acs.orglett.8b00302 Org. Lett. 2018, 20, 1802−1805
Letter
Organic Letters
Table 1. Parallel Experiments for Detecting 2 and Bra032185 Products by Yeast Heterologous Expressiona
a
Modified Folch extraction was done with 2:1 DCM/methanol. The ratio of epimers 2 to 1 in the expt I crude extract was determined from mean intensities of five pairs of resolved HSQC methyl signals. No signals for 2 were observed at a detection limit of 0.05% in expt II.
saponification was performed. These results suggest that 2 arose nonenzymatically in experiment I by epimerization of 1 via H4 deprotonation to the enolate, followed by reprotonation at C4 from below or above to give 1 or 2. Formation of 1 should be favored kinetically (by the 5β-methyl sterically hindering reprotonation from above) and thermodynamically (by generating an equatorial C4 substituent). DFT calculations (ΔG, B3PW91/6-311G(2d,p)) predicted a Boltzmann distribution of 3.0 and 4.4% 2 at 25 and 65 °C, respectively, relative to 1. Indeed, refluxing 1 (1 mg,
