Methionine synthases catalyze the formation of methionine by the transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine. This reaction is the last step in L-methionine biosynthesis, and it also serves to regenerate the methyl group of S-adenosylmethionine, a cofactor required for biological methylation reactions. We describe the cloning, expression and characterization of a methionine synthase from the higher plant Catharanthus roseus. cDNAs were identified that encoded a protein of 85 kDa sharing 50% identify with the cobalamin-independent methionine synthase from Escherichia coli (MetE) and 41% identity with a partial sequence of a yeast homolog of MetE. The C. roseus protein was expressed at high levels in E. coli. The enzyme accepts the triglutamate form of methyltetrahydrofolate as a methyl donor but not the monoglutamate form, and it does not require S-adenosylmethionine or cobalamin for activity. The properties indicate that the enzyme is a cobalamin-independent methionine synthase (EC 2.1.1.14). In contrast to the E. coli MetE, the plant protein does not require phosphate or magnesium ions for activity. Immunoblots of plants extracts showed that the protein was localized in the cytosol, and was present in a variety of plant species. A nutritional downshift of the C. roseus cell culture revealed a strong, transient transcriptional activation, but no significant increment in the total level of the protein. The availability of the protein and the cDNA now provide tools to investigate the complexities of methionine biosynthesis in plants.

Summary The molecular characterization of CYP72A1 from Catharanthus roseus (Madagascar periwinkle) was described nearly a decade ago, but the enzyme function remained unknown. We now show by in situ hybridization and immunohistochemistry that the expression in immature leaves is epidermis‐specific. It thus follows the pattern previously established for early enzymes in the pathway to indole alkaloids, suggesting that CYP72A1 may be involved in their biosynthesis. The early reactions in that pathway, i.e. from geraniol to strictosidine, contain several candidates for P450 activities. We investigated in this work two reactions, the conversion of 7‐deoxyloganin to loganin (deoxyloganin 7‐hydroxylase, DL7H) and the oxidative ring cleavage converting loganin into secologanin (secologanin synthase, SLS). The action of DL7H has not been demonstrated in vitro previously, and SLS has only recently been identified as P450 activity in one other plant. We show for the first time that both enzyme activities are present in microsomes from C. roseus cell cultures. We then tested whether CYP72A1 expressed in E. coli as a translational fusion with the C. roseus P450 reductase (P450Red) has one or both of these activities. The results show that CYP72A1 converts loganin into secologanin.

A PCR-based approach was used to isolate cDNAs for cinnamate 4-hydroxylase (C4H) from Catharanthus roseus cell cultures. The protein shared 75.9% identity with C4H from other plants, and the transcription was induced under various stress conditions. The cloned protein was used to investigate the functional expression of plant P45o/P45o-reductase fusions in E. coli. Fusions containing a modified N-terminal membrane anchor were located in the membrane and possessed C4H activity without solubilization or addition of other factors. The results indicate that the fusion protein strategy provides a useful tool to analyze the activities encoded in the rapidly increasing number of plant P45o sequences of uncertain or unknown function. We also discuss critical elements of the strategy: the choice of the E. coli host strain, the N-terminal membrane anchor, and the conditions for protein expression.

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Methionine synthases catalyze the formation of methionine by the transfer of a methyl group from 5‐methyltetrahydrofolate to homocysteine. This reaction is the last step in l‐methionine biosynthesis, and it also serves to regenerate the methyl group of 5‐adenosylmethionine, a cofactor required for biological methylation reactions. We describe the cloning, expression and characterization of a methionine synthase from the higher plant Catharanthus roseus. cDNAs were identified that encoded a protein of 85 kDa sharing 50 % identity with the cobalamin‐independent methionine synthase from Escherichia coli (MetE) and 41 % identity with a partial sequence of a yeast homolog of MetE. The C. roseus protein was expressed at high levels in E. coli. The enzyme accepts the triglutamate form of methyltetrahydrofolate as a methyl donor but not the monoglutamate form, and it does not require 5‐adenosylmethionine or cobalamin for activity. The properties indicate that the enzyme is a cobalamin‐independent methionine synthase (EC 2.1.1.14). In contrast to the E. coli MetE, the plant protein does not require phosphate or magnesium ions for activity. Immunoblots of plant extracts showed that the protein was localized in the cytosol, and was present in a variety of plant species. A nutritional downshift of the C. roseus cell culture revealed a strong, transient transcriptional activation, but no significant increment in the total level of the protein. The availability of the protein and the cDNA now provide tools to investigate the complexities of methionine biosynthesis in plants.