Toni M. Kutchan

The Pseudomonas phytotoxin coronatine mimics octadecanoid signalling molecules of higher plants

The phytotoxic principle, coronatine, which is present in several pathovars of the plant pathogen, Pseudomonas syringae was shown to be highly active in completely different, jasmonate‐selective bioassays. At nanomolar to micromolar concentrations, coronatine induced the accumulation of defense‐related secondary metabolites in several plant cell cultures, induced transcript accumulation of the elicitor‐responsive gene encoding the berberine bridge enzyme of Eschscholtzia californica, as well as the coiling response of Bryonia dioica tendrils. Biological activity critically depended upon the structure of coronatine, and slight modifications, such as methylation of the carboxyl moiety or reduction of the carbonyl group, rendered the molecules almost inactive. Coronafacic acid, obtained by hydrolysis of coronatine, was also nearly inactive. Coronatine did not elicit the accumulation of endogenous jasmonic acid in the systems analyzed. While coronafacic acid is similar in structure to jasmonic acid, we found coronatine to be a close structural analogue of the cyclic C18‐precursor of jasmonic acid, 12‐oxo‐phytodienoic acid. The phytotoxic symptoms produced by coronatine can now be understood on the basis of the toxins action as a mimic of the octadecanoid signalling molecules of higher plants.

Enzymology and Molecular Biology of Aflatoxin Biosynthesis.

Abstract The biosynthetic pathways leading to the benzophenanthridine alkaloid, sanguinarine, and the indole alkaloids, ajmalicine and ajmaline, have been solved at the level of the individual enzymes involved. The cDNA for two of the key enzymes has been cloned and the enzymes subsequently actively expressed in heterologous organisms.

The cDNA clone for strictosidine synthase from Rauvolfia serpentina. DNA sequence determination and expression in Escherichia coli

The cDNA clone for strictosidine synthase, the enzyme which catalyzes the stereospecific condensation of tryptamine with secologanin to form the key intermediate in indole alkaloid biosynthesis, strictosidine, has been identified with a synthetic oligodeoxynucleotide hybridization probe in a λgt11 cDNA library of cultured cells of Rauvolfia serpentina. The DNA has been sequenced, revealing an open reading frame of 1032 base pairs encoding 344 amino acids. The sequence of 60 nucleotides in the 5′‐flanking region has been determined by primer extension analysis. The encoded protein has been expressed in E. coli DH5 as detected by immunoblotting of protein extracts with antibodies raised against the native enzyme.

Characterization and mechanism of the berberine bridge enzyme, a covalently flavinylated oxidase of benzophenanthridine alkaloid biosynthesis in plants.

The berberine bridge enzyme ((S)-reticuline:oxygen oxidoreductase (methylene-bridge-forming), EC 1.5.3.9) catalyzes the oxidative cyclization of the N-methyl moiety of (S)-reticuline into the berberine bridge carbon, C-8, of (S)-scoulerine. This is a reaction that has neither an equivalent in organic chemistry nor a parallel in nature. The uniqueness of this catalytic reaction prompted an in depth study that began with the isolation of the cDNA encoding the berberine bridge enzyme followed by the overexpression of this cDNA in insect cell culture. The heterologously expressed enzyme has herein been shown to contain covalently attached FAD in a molar ratio of cofactor to protein of 1:1.03. Site-directed mutagenesis and laser desorption time-of-flight mass spectrometry suggest that the site of covalent attachment is at His-104. The holoenzyme exhibited absorbance maxima at 380 and 442 nm and a fluorescence emission maximum at 628 nm (310 nm excitation). Enzymic transformation of a series of (S)-reticuline derivatives modified with respect to the stereochemistry at C-1 or in the aromatic ring substitution suggests that ring closure proceeds in two steps: formation of the methylene iminium ion and subsequent ring closure via an ionic mechanism.

Strictosidine: From alkaloid to enzyme to gene

In this review, the elucidation of the structure of the first key alkaloidal intermediate in monoterpenoid indole alkaloid biosynthesis, 3 alpha(S)-strictosidine, is presented. The discovery of the enzyme which catalyses the stereospecific formation of this alkaloidal precursor from tryptamine and secologanin, strictosidine synthase, is also detailed. From the knowledge provided by the stereochemical structure of strictosidine and the biochemical characteristics of the biosynthetic enzyme, strictosidine synthase, a new approach to the study of monoterpenoid indole alkaloid biosynthesis was developed. Physiological studies of monoterpenoid indole alkaloid biosynthesis at the enzymic level in plants and plant cell cultures were performed followed by the analyses of these systems at the level of molecular genetics.

The jasmonate precursor, 12‐oxo‐phytodienoic acid. Induces phytoalexin synthesis in Petroselinum crispum cell cultures

The pentacyclic biosynthetic precursor of jasmonic acid, 12‐oxo‐phytodienoic acid, was found to induce synthesis of the major flavonoid, apiin. in cell suspension cultures of Petroselinum crispum. The accumulation of apiin was preceded by an increase in the relative levels of poly (A)* RNAs that code for the flavonoid biosynthetic enzymes phenylalanine ammonia lyase, 4‐coumarate:CoA ligase and chalcone synthase, Poly (A)* RNAs reached maximal levels at approximately 4–6 h after the addition of elicitor while flavonoids continued to accumulate in the cultures for at least 6 days. 12‐Oxo‐phytodienoic acid is the first pentacyclic precursor in the jasmonic acid biosynthetic chain which functions as a signal transducer for phytoalexin induction.

Expression of enzymatically active cloned strictosidine synthase from the higher plant Rauvolfia serpentina in Escherichia coli

Strictosidine synthase; Indole alkaloid biosynthesis; Cloned enzyme expression; (Rauvolfia serpentina, Escherichia coli)

Saccharomyces cerevisiae and Neurospora crassa contain heavy metal sequestering phytochelatin

In fungi, cellular resistance to heavy metal cytotoxicity is mediated either by binding of metal ions to proteins of the metallothionein type or by chelation to phytochelatin-peptides of the general formula (γ-Glu-Cys)n-Gly. Hitherto, only one fungus, Candida glabrata has been shown to contain both metal inactivating systems. Here we show by unambiguous FAB-MS analysis that both a metallothionein-free mutant of Saccharomyces cerevisiae as well as a wildtype strain synthesize phytochelatin (PC2) upon exposure to 250 μM Cd2+ ions. The presence of Zn and/or Cu ions in the nutrient broth also induces PC2 synthesis in this organism. By 109Cd exchange and subsequent monobromobimane fluorescence HPLC, it could be shown that the presence of Cd2+ in the growth medium also induces phytochelatin synthesis in Neurospora crassa, which contains metallothioneins.

Heterologous expression of the plant proteins strictosidine synthase and berberine bridge enzyme in insect cell culture

The heterologous expression of cDNAs encoding the alkaloid biosynthetic enzymes, strictosidine synthase [EC 4.3.3.2] from Rauvolfia serpentina and the berberine bridge enzyme [(S)-reticuline: oxygen oxidoreductase (methylene-bridge-forming), EC 1.5.3.9] from Eschscholtzia californica, has been achieved in a cell culture (Sf9) of the fall army worm, Spodoptera frugiperda, using a baculovirus-based expression system. The expression resulted in the overproduction of each plant enzyme in a catalytically active form. The maximal production attained was 4 mg purified, active enzyme per litre cell culture for both the strictosidine synthase and berberine bridge enzymes.

(S)-norcoclaurine is the central intermediate in benzylisoquinoline alkaloid biosynthesis

Abstract Feeding experiments with ( S )-[1- 13 C]-norcoclaurine demonstrate that this trihydroxylated precursor is specifically incorporated into protoberberine, aporphine and benzophenanthridine alkaloids in cell suspension cultures, as well as into pavine and benzophenanthridine alkaloids in whole plants. The rates of incorporation ranged from 2.5 to 36%. This reveals that tyrosine is metabolized to dopamine and p -hydroxyphenylacetaldehyde which then condense to form norcoclaurine, thus explaining the lack of incorporation of DOPA or dopamine into the benzylic portion of reticuline derived alkaloids.