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. 2018 May 4;19(9):940-948.
doi: 10.1002/cbic.201700621. Epub 2018 Mar 22.

Discovery of a Short-Chain Dehydrogenase from Catharanthus roseus that Produces a New Monoterpene Indole Alkaloid

Anna K Stavrinides  1   2 , Evangelos C Tatsis  1 , Thu-Thuy Dang  1 , Lorenzo Caputi  1 , Clare E M Stevenson  1 , David M Lawson  1 , Bernd Schneider  3 , Sarah E O'Connor  1
Affiliations

Discovery of a Short-Chain Dehydrogenase from Catharanthus roseus that Produces a New Monoterpene Indole Alkaloid

Anna K Stavrinides et al. Chembiochem. .

Abstract

Plant monoterpene indole alkaloids, a large class of natural products, derive from the biosynthetic intermediate strictosidine aglycone. Strictosidine aglycone, which can exist as a variety of isomers, can be reduced to form numerous different structures. We have discovered a short-chain alcohol dehydrogenase (SDR) from plant producers of monoterpene indole alkaloids (Catharanthus roseus and Rauvolfia serpentina) that reduce strictosidine aglycone and produce an alkaloid that does not correspond to any previously reported compound. Here we report the structural characterization of this product, which we have named vitrosamine, as well as the crystal structure of the SDR. This discovery highlights the structural versatility of the strictosidine aglycone biosynthetic intermediate and expands the range of enzymatic reactions that SDRs can catalyse. This discovery further highlights how a sequence-based gene mining discovery approach in plants can reveal cryptic chemistry that would not be uncovered by classical natural product chemistry approaches.

Keywords: alkaloids; biosynthesis; natural products; reduction; short-chain dehydrogenases.

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Figures

Scheme 1
Scheme 1
Reactions of SDRs in specialized metabolism. A) Previously identified SDRs in specialized metabolism include iridoid synthase (ISY), tropinone reductases I and II (TRI and TRII), salutaridine reductase (SalR), menthone reductase (MNMR) and progesterone 5‐β‐reductase (PβR). B) Strictosidine aglycone is a central biosynthetic intermediate of MIA biosynthesis that can undergo reduction to form various alkaloid backbones. THAS1 and GS are previously discovered medium‐chain alcohol dehydrogenases.
Figure 1
Figure 1
NMR spectra (700 MHz, CDCl3) of VAS (Cro013448) product. Top left and top right: sections of the 1H NMR spectrum. Bottom left: ROESY. Bottom right: HMBC. Strictosidine and vallesiachotamine numbering. The signal of 18‐CH3 (δ H=1.36 ppm) is not included in this figure. Ring D is indicated on the proposed chemical structure.
Scheme 2
Scheme 2
Rearrangement of strictosidine aglycone to give rise to different MIA structural classes. Rotation around the C‐15−C‐20 bond (blue arrow) and subsequent cyclization of ring D give rise to the heteroyohimbine and geissoschizine backbones. Rotation around the C‐14−C‐15 bond (red arrow) and subsequent cyclization of ring D yields the substrate of Cro013448, leading to vallesiachotamine (15), antirhine (16, previously observed natural products) and vitrosamine (4).
Figure 2
Figure 2
NMR spectra of deuterated product of VAS (Cro013448) (top) in comparison with non‐deuterated product (bottom). Signals from contaminants are coloured in grey.
Figure 3
Figure 3
X‐ray structure of C. roseus vitrosamine synthase (VAS). A), B) Orthogonal cartoon representations of VAS, in which the core structure is coloured in magnolia and the flap domain and cofactor loop are shown in red and cyan, respectively. The black spheres indicate where there is a break in the backbone trace of the flap domain corresponding to a region that could not be resolved in the electron density. Also shown as van der Waals spheres are the bound NADP+ cofactor (light brown with C‐4 in yellow) and the docked substrate (ice blue). Additionally, in B), the flap domains of close structural homologues are shown after superposition of their full structures onto that of VAS. Specifically, these are P. somniferum SalR (pink; PDB ID: 3O26), M. piperita MNMR (green; PDB ID: 5L53) and human CBR1 (blue; PDB ID: 1WMA). However, in the last case the flap domain is merely a short loop connecting the equivalent of β4 and α4 in VAS. The full structural superposition is shown as a stereoview in Figure S10. C), D) The region highlighted in A) with the protein depicted as a molecular surface, with and without the docked substrate, respectively. Note that the cofactor loop almost entirely covers the nicotinamide “half” of the NADP+, except for the outer edge of the nicotinamide ring bearing C‐4, and that this is occluded by the docked substrate, which closely matches the dimensions of the active‐site pocket. E) The same region as D), but from above with the protein in cartoon. The NADP+ is coloured green (carbon atoms) but with the nicotinamide C‐4 atom in yellow. Also shown is the canonical SDR catalytic triad of Ser167, Tyr223 and Lys227, together with 1.55 Å resolution omit difference electron density for the cofactor (contoured at ≈5.0 σ). In this view, part of the flap domain was omitted for clarity.
Figure 4
Figure 4
Biochemical assay of VAS. A) LC‐MS chromatograms showing product formation of VAS wild type (Cro013448, red) and its mutants Y223A (orange), S167A (blue) and K227A (green). LC‐MS chromatograms of the hydrated (371 m/z) and the dehydrated product (353 m/z) on the left and right, respectively. B) Typical spectrum of NADPH absorbance at 340 nm during reduction of strictosidine aglycone (100 μm) by THAS1 and VAS (Cro013448) at pH 6. The addition of VAS was after THAS1 had reduced its available substrate (plateau). VAS further reduced strictosidine aglycone, thus indicating that these two enzymes do not compete for the same substrate.
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