Molecular and biochemical analysis of a Madagascar periwinkle root-specific minovincinine-19-hydroxy-O-acetyltransferase

Abstract

The terminal steps in the biosynthesis of the monoterpenoid indole alkaloids vindoline and minovincinine are catalyzed by separate acetyl coenzyme A-dependent O-acetyltransferases in Madagascar periwinkle (Catharanthus roseus G. Don). Two genes were isolated that had 63% nucleic acid identity and whose deduced amino acid sequences were 78% identical. Active enzymes that were expressed as recombinant His-tagged proteins in Escherichia coli were named minovincinine-19-O-acetyltransferase (MAT) and deacetylvindoline-4-O-acetyltransferase (DAT) because they catalyzed the 19-O-acetylation of indole alkaloids such as minovincinine and hörhammericine and the 4-O-acetylation of deacetylvindoline, respectively. Kinetic studies showed that the catalytic efficiency of recombinant MAT (rMAT) was very poor compared with that of recombinant DAT (rDAT), whose turnover rates for Acetyl-coenzyme A and deacetylvindoline were approximately 240- and 10,000-fold greater than those of rMAT. Northern-blot analyses showed that MAT is expressed in cortical cells of the root tip, whereas DAT is only expressed in specialized idioblast and laticifer cells within light exposed tissues like leaves and stems. The coincident expression of trytophan decarboxylase, strictosidine synthase, and MAT within root cortical cells suggests that the entire pathway for the biosynthesis of tabersonine and its substituted analogs occurs within these cells. The ability of MAT to catalyze the 4-O-acetylation of deacetylvindoline with low efficiency suggests that this enzyme, rather than DAT, is involved in vindoline biosynthesis within transformed cell and root cultures, which accumulate low levels of this alkaloid under certain circumstances.

Figure 1

Biosynthesis of tabersonine-derived indole alkaloids in Madagascar periwinkle organs. Tabersonine is converted into vindoline via six enzymatic steps, with the terminal hydroxylation (D4H) and O-acetylation (DAT) occurring in specialized cells known as idioblasts and laticifers of leaves and stems [1]. Tabersonine is converted into lochnericine, hörhammericine, and minovincinine via uncharacterized hydroxylations and 19-hydroxy-indole alkaloids are converted into their respective products by O-acetylation (MAT). These tabersonine analogs are known to accumulate under certain conditions within cell cultures (Kutney et al., 1980) and roots (Shanks et al., 1998) of Madagascar periwinkle.

Figure 2

Amino acid alignment of rMAT and rDAT gene products. Identical amino acids are shown in black, conserved substitutions are in white on a black background, and differing amino acids are gray. The boxed residues highlight the conserved HXXXDG active site and DFGWGKP motif and the arrow identifies the active site His residue.

Figure 3

Autoradiogram of reaction products obtained from MAT-catalyzed reactions. Lochnericine (lane 1), hörhammericine (lane 2), or a minovincinine-containing root extract (lane 3) were used as substrates. The reaction products were extracted and chromatographed as described in “Material and Methods.” The structures of the respective acetylated products are shown. Although no acetylated product was observed when the supplied substrate was lochnericine, its structure is included to show the requirement for the hydroxyl group at position 19 for enzyme activity.

Figure 4

Northern blots of total RNA isolated from Madagascar periwinkle hairy roots, roots, stems, leaves, flower petals, or etiolated seedlings, respectively. Blots were probed with the DAT ORF fragment and a 423-bp HindIII fragment of MAT::pBluescript under high stringency conditions. RNA was quantified by ethidium bromide staining and 20 μg of total RNA per sample was electrophoresed on a 7% (v/v) formaldehyde agarose gel. RNA was transferred to nitrocellulose for hybridization.

Figure 5

Northern blots of total RNA were isolated from the following: A, 0- to 7-d-old etiolated (D) seedlings, or 5-d-old etiolated seedlings treated with light (L) for of 24 h (6L); and B, 5-d-old whole etiolated seedlings (W), roots (R), hypocotyls (H), and cotyledons (C). Hybridization was carried out under high stringency conditions using a 423-bp HindIII fragment of MAT::pBluescript. RNA was quantified by ethidium bromide staining and 20 μg of total RNA was electrophoresed on a 7% (v/v) formaldehyde agarose gel and transferred to nitrocellulose for hybridization. The blots in A and B were exposed for 3 d, respectively. C, Distribution of MAT activity in 0- to 10-d-old radicles isolated from dark-grown seedlings. MAT activity was determined by liquid scintillation counting of radioactive echitovenine after isolation by TLC (see “Materials and Methods”). Data presented are the average of two trials.

Figure 6

Tissue- and cell-specific localization of mat. A, Northern blot of total RNA isolated from 0.5-cm sections of lateral hairy root tissue (as shown in the schematic). Hybridization with a 423-bp HindIII fragment of MAT::pBluescipt was carried out under high stringency conditions. B, Localization of mat mRNA by in situ RNA hybridization in hairy roots. The longitudinal section of a 14-d-old lateral hairy root apex was hybridized with antisense RNA for mat as described in “Materials and Methods.” Magnification = 250×. Bar = 100 μm.

Figure 7

Southern-blot analysis of the MAT and DAT genes in Madagascar periwinkle. A, Genomic DNA isolated from Madagascar periwinkle leaves was restriction digested with EcoRI (R), HindIII (H), XbaI (X), and EcoRV (V). Approximately 12 μg of genomic DNA was electrophoresed per lane, in duplicate, to allow for hybridization under high stringency conditions with the DAT ORF fragment and a 423-bp HindIII fragment of MAT::pBluescript, respectively. B, Southern blots of approximately 100 pg of MAT ORF and DAT ORF fragments illustrating the specificity of the probes used for hybridization. MAT (423-bp HindIII fragment of MAT::pBluescript)and DAT (the DAT ORF fragment). C, Restriction map of the respective genomic clones, gDAT (gDAT-6) and gMAT (gDAT-15), as deduced from the restriction digest patterns of the genomic Southerns. The ORF segments are illustrated as boxes, with arrows indicating the orientation of the ORF in the genomic clones.