On the evolution of alternate core packing in eightfold β/α-barrels

Two sequence-related subfamilies of flavin-binding β/α-barrels have been identified (the type I and type II proteins) that differ in the nature of residue packing in the core of the barrel domain. Similar observed differences in the packing of internal amino acid side chains in β/α-barrels have previously been used to argue that these domains have evolved convergently toward a stable structural framework. Using structural alignments of flavin-binding barrel proteins, we demonstrate that simple genetic alterations may be responsible for switching the nature of side-chain packing observed in β/α-barrels. The implication is that the 2 structural classes of β/α-barrel cores can arise divergently from an ancestral barrel framework and that convergent evolution to a stable fold need not be invoked to account for the emergence of 2 classes of β/α-barrel core.     

Differential inhibition of indole-3-acetic acid and tryptophan biosynthesis by indole analogues. I. Tryptophan dependent IAA biosynthesis

Maize liquid endosperm extracts contain the enzymes necessary for all of the steps of the plant IAA biosynthetic pathway from tryptophan, and provide a means to assay the pathway in vitro. We have analyzed the reactions in the presence of a series of indole and indole-like analogues in order to evaluate the potential of these compounds to act as inhibitors of IAA biosynthesis. Such inhibitors will be useful to investigate the tryptophan to IAA pathway, to determine the precursors and intermediates involved, and to select for mutants in this process. A number of such compounds were tested using in vitro enzyme assays for both the tryptophan dependent IAA biosynthesis pathway and for tryptophan synthase activity. Some compounds showed strong inhibition of IAA biosynthesis while having only a slight effect on the reaction rate of tryptophan synthase . These results: (1) show that IAA biosynthesis can be selectively inhibited relative to tryptophan biosynthesis; (2) suggest potential ways to screen for IAA biosynthetic pathway mutations in plants; and (3) provide additional tools for studies of IAA biosynthesis in plants.     

Free tryptophan pool and tryptophan biosynthetic enzymes in Saccharomyces cerevisiae

The free tryptophan pool and the levels of two enzymes of tryptophan biosynthesis (anthranilate synthase and indoleglycerolphosphate synthase) have been determined in a wild type strain of Saccharomyces cerevisiae and in mutants with altered regulatory properties.The tryptophan pool of wild type cells growing in minimal medium is 0.07 mole per g dry weight. Addition of anthranilate, indole or tryptophan to the medium produces a fifteen- to forty-fold increase in tryptophan pool, but causes no repression of the biosynthetic enzymes. Inclusion of 5-methyltryptophan in the growth medium causes a reduction in growth rate and a derepression of the biosynthetic enzymes, and this is shown here not to be correlated with a decrease in the free tryptophan pool.Mutants with an altered anthranilate synthase showing decreased sensitivity to inhibition by l-tryptophan or by the analogue dl-5-methyltryptophan have a tryptophan pool far higher than the wild type strain, but no repression of indoleglycerolphosphate synthase was observed. Mutants with an anthranilate synthase more sensitive to tryptophan inhibition show a slightly reduced tryptophan pool, but no derepression of indoleglycerolphosphate synthase was found.A mutant with constitutively derepressed levels of the biosynthetic enzymes shows a considerably increased tryptophan pool. Addition of 5-methyltryptophan to the growth medium of non-derepressible mutants causes a decrease in growth rate accompanied by a decrease in the tryptophan pool.Abbreviations CDRP 1-(o-carboxyphenylamino)-1-deoxyribulosephosphate - paba paraaminobenzoic acid - PRA N-(5-phosphoribosyl)-anthranilate - tRNA transfer ribonucleic acid; trp1 to trp5 refer to the structural genes for corresponding tryptophan biosynthetic enzymes     

Evolutionarily Conserved Optimization of Amino Acid Biosynthesis

The “cognate bias hypothesis” states that early in evolutionary history the biosynthetic enzymes for amino acid x gradually lost residues of x, thereby reducing the threshold for deleterious effects of x scarcity. The resulting reduction in cognate amino acid composition of the enzymes comprising a particular amino acid biosynthetic pathway is predicted to confer a selective growth advantage on cells. Bioinformatic evidence from protein-sequence data of two bacterial species previously demonstrated reduced cognate bias in amino acid biosynthetic pathways. Here we show that cognate bias in amino acid biosynthesis is present in the other domains of life—Archaebacteria and Eukaryota. We also observe evolutionarily conserved underrepresentations (e.g., glycine in methionine biosynthesis) and overrepresentations (e.g., tryptophan in asparagine biosynthesis) of amino acids in noncognate biosynthetic pathways, which can be explained by secondary amino acid metabolism. Additionally, we experimentally validate the cognate bias hypothesis using the yeast Saccharomyces cerevisiae. Specifically, we show that the degree to which growth declines following amino acid deprivation is negatively correlated with the degree to which an amino acid is underrepresented in the enzymes that comprise its cognate biosynthetic pathway. Moreover, we demonstrate that cognate fold representation is more predictive of growth advantage than a host of other potential growth-limiting factors, including an amino acid’s metabolic cost or its intracellular concentration and compartmental distribution. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. Reviewing Editor: Dr. Niles Lehman Ethan O. Perlstein and Benjamin L. de Bivort contributed equally to this work.     

Cross-Pathway Regulation: Histidine-Mediated Control of Histidine, Tryptophan, and Arginine Biosynthetic Enzymes in Neurospora crassa

In Neurospora crassa, histidine starvation of histidine mutants resulted in derepression of histidine, tryptophan, and arginine biosynthetic enzymes. The same tripartite derepression occurred in wild-type strain 74A when it was grown in medium supplemented with 3-amino-1,2,4-triazole, an inhibitor of histidine biosynthesis. Histidine-mediated derepression of tryptophan and arginine biosynthetic enzymes was not due to a lowered intracellular concentration of tryptophan or arginine, respectively. A discussion of possible mechanisms and of similar studies in prokaryotic and eukaryotic organisms is presented.     

拟南芥色氨酸与吲哚乙酸生物合成的研究进展

拟南芥色氨酸生物合成途径的研究已逐渐成为植物分子生物学家了解植物基因结构和表达调控最主要的模式系统之一。到目前为止,编码拟南芥色氨酸合成途径的七种酶蛋白的基因已经全部被克隆,并进行了不同程度的分子生物学研究。长期以来,色氨酸一直被认为是植物生长素吲哚乙酸（ＩＡＡ）生物合成（从头合成）的前体物,但近年来人们发现生长素合成的非色氨酸途径可能是其在植物中生物合成的主要途径。植物在不同的发育阶段可能采用不同的方式合成ＩＡＡ。     

Regulation of tryptophan metabolism in Coprinus cinereus: Isolation and characterisation of mutants resistant to 5-fluoroindole

171 mutations conferring resistance to the indole analogue 5-fluoroindole (5 FI) were isolated in the filamentous basidiomycete fungus Coprinus cinereus. 5 FI is thought to be toxic because it is converted intracellularly to 5-fluorotryptophan (5 FT) which feedback inhibits the first enzyme of the tryptophan biosynthetic pathway, anthranilate synthase. Mutations were assigned to five loci, iar-1-iar-5 on the basis of functional analyses and mapping experiments. iar-5 mutations mapped in the anthranilate synthase structural gene and gave rise to an enzyme feedback resistant to tryptophan and its analogue. Mutants at other loci had regulatory changes. iar-1 and iar-3 mutants had elevated levels of two pathway enzymes measured (anthranilate synthase and tryptophan synthase) and were cross resistant to analogues of other aromatic amino acids suggesting that the entire aromatic pathway was derepressed. iar-3 mutants were unable to degrade metabolically derived typtophan to anthranilic acid unlike iar-1 mutants which excreted high levels of anthranilic acid. iar-2 mutants appeared to have a constitutive degradative pathway. iar-4 mutants had a blocked degradative pathway and unusual levels of tryptophan pathway enzymes.Abbreviations 5 FI 5-fluoroindole - 5 FT 5-fluorotryptophan - pFP para-fluorophenylalanine - mFT meta-fluoro-tyrosine     

Evidence for the thiamine biosynthetic pathway in higher-plant plastids and its developmental regulation

Thiamine or vitamin B-1, is an essential constituent of all cells since it is a cofactor for two enzyme complexes involved in the citric acid cycle, pyruvate dehydrogenase and -ketoglutarate dehydrogenase. Thiamine is synthesized by plants, but it is a dietary requirement for humans and other animals. The biosynthetic pathway for thiamine in plants has not been well characterized and none of the enzymes involved have been isolated. Here we report the cloning and characterization of two cDNAs representing members of the maize thi1 gene family encoding an enzyme of the thiamine biosynthetic pathway. This assignment was made based on sequence homology to a yeast thiamine biosynthetic gene and by functional complementation of a yeast strain in which the endogenous gene was inactivated. Using immunoblot analysis, the thi1 gene product was found to be located in a plastid membrane fraction. RNA gel blot analysis of various tissues and developmental stages indicated thi1 expression was differentially regulated in a manner consistent with what is known about thiamine synthesis in plants. This is the first report of cDNAs encoding proteins involved in thiamine biosynthesis for any plant species.     

Loop‐loop interactions govern multiple steps in indole‐3‐glycerol phosphate synthase catalysis

Substrate binding, product release, and likely chemical catalysis in the tryptophan biosynthetic enzyme indole‐3‐glycerol phosphate synthase (IGPS) are dependent on the structural dynamics of the β1α1 active‐site loop. Statistical coupling analysis and molecular dynamic simulations had previously indicated that covarying residues in the β1α1 and β2α2 loops, corresponding to Arg54 and Asn90, respectively, in the Sulfolobus sulfataricus enzyme (ssIGPS), are likely important for coordinating functional motions of these loops. To test this hypothesis, we characterized site mutants at these positions for changes in catalytic function, protein stability and structural dynamics for the thermophilic ssIGPS enzyme. Although there were only modest changes in the overall steady‐state kinetic parameters, solvent viscosity and solvent deuterium kinetic isotope effects indicated that these amino acid substitutions change the identity of the rate‐determining step across multiple temperatures. Surprisingly, the N90A substitution had a dramatic effect on the general acid/base catalysis of the dehydration step, as indicated by the loss of the descending limb in the pH rate profile, which we had previously assigned to Lys53 on the β1α1 loop. These changes in enzyme function are accompanied with a quenching of ps‐ns and µs‐ms timescale motions in the β1α1 loop as measured by nuclear magnetic resonance studies. Altogether, our studies provide structural, dynamic and functional rationales for the coevolution of residues on the β1α1 and β2α2 loops, and highlight the multiple roles that the β1α1 loop plays in IGPS catalysis. Thus, substitution of covarying residues in the active‐site β1α1 and β2α2 loops of indole‐3‐glycerol phosphate synthase results in functional, structural, and dynamic changes, highlighting the multiple roles that the β1α1 loop plays in enzyme catalysis and the importance of regulating the structural dynamics of this loop through noncovalent interactions with nearby structural elements.     

Structural and functional studies of a trans‐acyltransferase polyketide assembly line enzyme that catalyzes stereoselective α‐ and β‐ketoreduction

While the cis‐acyltransferase modular polyketide synthase assembly lines have largely been structurally dissected, enzymes from within the recently discovered trans‐acyltransferase polyketide synthase assembly lines are just starting to be observed crystallographically. Here we examine the ketoreductase (KR) from the first polyketide synthase module of the bacillaene nonribosomal peptide synthetase/polyketide synthase at 2.35‐Å resolution. This KR naturally reduces both α‐ and β‐keto groups and is the only KR known to do so during the biosynthesis of a polyketide. The isolated KR not only reduced an N‐acetylcysteamine‐bound β‐keto substrate to a D ‐β‐hydroxy product, but also an N‐acetylcysteamine‐bound α‐keto substrate to an L ‐α‐hydroxy product. That the substrates must enter the active site from opposite directions to generate these stereochemistries suggests that the acyl‐phosphopantetheine moiety is capable of accessing very different conformations despite being anchored to a serine residue of a docked acyl carrier protein. The features enabling stereocontrolled α‐ketoreduction may not be extensive since a KR that naturally reduces a β‐keto group within a cis‐acyltransferase polyketide synthase was identified that performs a completely stereoselective reduction of the same α‐keto substrate to generate the D ‐α‐hydroxy product. A sequence analysis of trans‐acyltransferase KRs reveals that a single residue, rather than a three‐residue motif found in cis‐acyltransferase KRs, is predictive of the orientation of the resulting β‐hydroxyl group. Proteins 2014; 82:2067–2077. © 2014 Wiley Periodicals, Inc.     

Vitamin B1 de novo synthesis in the human malaria parasite Plasmodium falciparum depends on external provision of 4-amino-5-hydroxymethyl-2-methylpyrimidine

Vitamin B1 (thiamine) is an essential cofactor for several key enzymes of carbohydrate metabolism. Mammals have to salvage this crucial nutrient from their diet to complement their deficiency of de novo synthesis. In contrast, bacteria, fungi, plants and, as reported here, Plasmodium falciparum, possess a vitamin B1 biosynthesis pathway. The plasmodial pathway identified consists of the three vitamin B1 biosynthetic enzymes 5-(2-hydroxy-ethyl)-4-methylthiazole (THZ) kinase (ThiM), 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP)/HMP-P kinase (ThiD) and thiamine phosphate synthase (ThiE). Recombinant PfThiM and PfThiD proteins were biochemically characterised, revealing K(m)app values of 68 microM for THZ and 12 microM for HMP. Furthermore, the ability of PfThiE for generating vitamin B1 was analysed by a complementation assay with thiE-negative E. coli mutants. All three enzymes are expressed throughout the developmental blood stages, as shown by Northern blotting, which indicates the presence of the vitamin B1 biosynthesis enzymes. However, cultivation of the parasite in minimal medium showed a dependency on the provision of HMP or thiamine. These results demonstrate that the human malaria parasite P. falciparum possesses active vitamin B1 biosynthesis, which depends on external provision of thiamine precursors.     

Applications of Biocatalysis to Biotechnology

β-lactam antibiotics (e.g. penicillins, cephalosporins) are of major clinical importance and contribute to over 40% of the total antibiotic market. These compounds are produced as secondary metabolites by certain actinomycetes and filamentous fungi (e.g. Penicillium, Aspergillus and Acremonium species). The industrial producer of penicillin is the fungus Penicillium chrysogenum. The enzymes of the penicillin biosynthetic pathway are well characterized and most of them are encoded by genes that are organized in a cluster in the genome. Remarkably, the penicillin biosynthetic pathway is compartmentalized: the initial steps of penicillin biosynthesis are catalyzed by cytosolic enzymes, whereas the two final steps involve peroxisomal enzymes. Here, we describe the biochemical properties of the enzymes of β-lactam biosynthesis in P. chrysogenum and the role of peroxisomes in this process. An overview is given     

Photosynthetic generation of heterologous terpenoids in cyanobacteria

The work aims to convert the secondary slow metabolism of the terpenoid biosynthetic pathway into a primary activity in cyanobacteria and to generate heterologous products using these photosynthetic microorganisms as cell factories. Case study is the production of the 10-carbon monoterpene β-phellandrene (PHL) in Synechocystis sp. PCC 6803 (Synechocystis). Barriers to this objective include the slow catalytic activity of the terpenoid metabolism enzymes that limit rates and yield of product synthesis and accumulation. “Fusion constructs as protein overexpression vectors” were applied in the overexpression of the geranyl diphosphate synthase (GPPS) and β-phellandrene synthase (PHLS) genes, causing accumulation of GPPS up to 4% and PHLS up to 10% of the total cellular protein. Such GPPS and PHLS protein overexpression compensated for their slow catalytic activity and enabled transformant Synechocystis to constitutively generate 24 mg of PHL per g biomass (2.4% PHL:biomass, w-w), a substantial improvement over earlier yields. The work showed that a systematic overexpression, at the protein level, of the terpenoid biosynthetic pathway genes is a promising approach to achieving high yields of prenyl product biosynthesis, on the way to exploiting the cellular terpenoid metabolism for commodity product generation.     

Metabolic flux in both the purine mononucleotide and histidine biosynthetic pathways can influence synthesis of the hydroxymethyl pyrimidine moiety of thiamine in Salmonella enterica

Together, the biosyntheses of histidine, purines, and thiamine pyrophosphate (TPP) contain examples of convergent, divergent, and regulatory pathway integration. Mutations in two purine biosynthetic genes (purI and purH) affect TPP biosynthesis due to flux through the purine and histidine pathways. The molecular genetic characterization of purI mutants and their respective pseudorevertants resulted in the conclusion that <1% of the wild-type activity of the PurI enzyme was sufficient for thiamine but not for purine synthesis. The respective pseudorevertants were found to be informational suppressors. In addition, it was shown that accumulation of the purine intermediate aminoimidazole carboxamide ribotide inhibits thiamine synthesis, specifically affecting the conversion of aminoimidazole ribotide to hydroxymethyl pyrimidine.     

Physiological and enzymatic aspects of histidine-mediated control of the tryptophan pathway

Summary It would thus appear that in Saccharomyces cerevisiae there are two forms of histidine-mediated control on the tryptophan pathway. In some strains histidine increases anthranilate synthetase and indole glycerol phosphate synthetase activities, while tryptophan synthetase decreases. In other strains histidine affects coordinately all enzymatic activities involved in tryptophan biosynthesis. The two groups of strains also differ in the formation, during the growth of the enzymatic activities involved in tryptophan biosynthesis. This difference in the relative rates at which the two enzymes are formed may explain the accumulation of intermediates in the cultural media of some strains. The derepression of anthranilate synthetase and indole glycerol phosphate synthetase activities by histidine is particularly manifest in the auxotrophic his₃ strains that show these activities very depressed in histidine starvation; large amounts of this amino acid stimulate them to a considerably greater extent than in prototrophic strains.Abbreviations IGP imidazole glycerol phosphate - InGP indole glycerol phosphate - ASase anthranilate synthetase - InGPase indole-3-glycerol phosphate synthetase - TSase tryptophan synthetase - Tris tris (hydroxymethyl)-aminomethane This investigation was supported by a research grant of C.N.R. (Consiglio Nazionale delle Ricerche, Roma).     

Induction of Arabidopsis tryptophan pathway enzymes and camalexin by amino acid starvation, oxidative stress, and an abiotic elicitor.

The tryptophan (Trp) biosynthetic pathway leads to the production of many secondary metabolites with diverse functions, and its regulation is predicted to respond to the needs for both protein synthesis and secondary metabolism. We have tested the response of the Trp pathway enzymes and three other amino acid biosynthetic enzymes to starvation for aromatic amino acids, branched-chain amino acids, or methionine. The Trp pathway enzymes and cytosolic glutamine synthetase were induced under all of the amino acid starvation test conditions, whereas methionine synthase and acetolactate synthase were not. The mRNAs for two stress-inducible enzymes unrelated to amino acid biosynthesis and accumulation of the indolic phytoalexin camalexin were also induced by amino acid starvation. These results suggest that regulation of the Trp pathway enzymes under amino acid deprivation conditions is largely a stress response to allow for increased biosynthesis of secondary metabolites. Consistent with this hypothesis, treatments with the oxidative stress-inducing herbicide acifluorfen and the abiotic elicitor alpha-amino butyric acid induced responses similar to those induced by the amino acid starvation treatments. The role of salicylic acid in herbicide-mediated Trp and camalexin induction was investigated.     

Molecular evolution of the histidine biosynthetic pathway

The available sequences of genes encoding the enzymes associated with histidine biosynthesis suggest that this is an ancient metabolic pathway that was assembled prior to the diversification of the Bacteria, Archaea, and Eucarya. Paralogous duplications, gene elongation, and fusion events involving different his genes have played a major role in shaping this biosynthetic route. Evidence that the hisA and the hisF genes and their homologues are the result of two successive duplication events that apparently took place before the separation of the three cellular lineages is extended. These two successive gene duplication events as well as the homology between the hisH genes and the sequences encoding the TrpG-type amidotransferases support the idea that during the early stages of metabolic evolution at least parts of the histidine biosynthetic pathway were mediated by enzymes of broader substrate specificities. Maximum likelihood trees calculated for the available sequences of genes encoding these enzymes have been obtained. Their topologies support the possibility of an evolutionary proximity of archaebacteria with low GC Gram-positive bacteria. This observation is consistent with those detected by other workers using the sequences of heat-shock proteins (HSP70), glutamine synthetases, glutamate dehydrogenases, and carbamoylphosphate synthetases.Abbreviations as amino acid - ORF open reading frame - bp base pair - kb 10³ bp - CarA carbamoyl phosphate synthetase (EC 6.3.5.5) - GAT glutamine amidotransferase - GuaA GMP synthetase (EC 6.3.4.1) - PabA 4-amino-4-deoxychorismate synthase (EC 4.1.3-) - PyrG GTP synthetase (EC 6.3.4.2) - AICAR 5-aminoimidazole-4-carboxamide-l--d ribofuranosyl 5-monophosphate - HAL l-histidinal - HOL l-histidinol - HP histidinol phosphate - IAP imidazole acetol-phosphate - IGP imidazole glycerol phosphate - PR phosphoribosyl - PRFAR N-[(5-phosphoribulosyl) formimino]-5-aminoimidazole-4-carboxamide ribonucleotide - 5-ProFAR N ¹-[(5-phosphoribosyl) formimino]-5-aminoimidazole-4-carboxamide ribonucleotide - PRPP phosphoribosyl-pyrophosphate - RFLP restriction fragment length polymorphism Correspondence to: R. Fani     

Experimental and computational analysis of the ancestry of an evolutionary young enzyme from histidine biosynthesis

The conservation of fold and chemistry of the enzymes associated with histidine biosynthesis suggests that this pathway evolved prior to the diversification of Bacteria, Archaea, and Eukaryotes. The only exception is the histidinol phosphate phosphatase (HolPase). So far, non-homologous HolPases that possess distinct folds and belong to three different protein superfamilies have been identified in various phylogenetic clades. However, their evolution has remained unknown to date. Here, we analyzed the evolutionary history of the HolPase from γ-Proteobacteria (HisB-N). It has been argued that HisB-N and its closest homologue d -glycero-d -manno-heptose-1,7-bisphosphate 7-phosphatase (GmhB) have emerged from the same promiscuous ancestral phosphatase. GmhB variants catalyze the hydrolysis of the anomeric d -glycero-d -manno-heptose-1,7-bisphosphate (αHBP or βHBP) with a strong preference for one anomer (αGmhB or βGmhB). We found that HisB-N from Escherichia coli shows promiscuous activity for βHBP but not αHBP, while βGmhB from Crassaminicella sp. shows promiscuous activity for HolP. Accordingly, a combined phylogenetic tree of αGmhBs, βGmhBs, and HisB-N sequences revealed that HisB-Ns form a compact subcluster derived from βGmhBs. Ancestral sequence reconstruction and in vitro analysis revealed a promiscuous HolPase activity in the resurrected enzymes prior to functional divergence of the successors. The following increase in catalytic efficiency of the HolP turnover is reflected in the shape and electrostatics of the active site predicted by AlphaFold. An analysis of the phylogenetic tree led to a revised evolutionary model that proposes the horizontal gene transfer of a promiscuous βGmhB from δ- to γ-Proteobacteria where it evolved to the modern HisB-N.     

Secondary metabolite biosynthesis in cultured cells of Catharanthus roseus (L.) G. Don immobilized by adhesion to glass fibres

Summary Suspension-cultured cells of Catharanthus roseus (L.) G. Don were immobilized on glass fibre mats and cultivated in shake flasks. The highly-aggregated immobilized cells exhibited a slower growth rate and accumulated reduced levels of tryptamine and indole alkaloids, represented by catharanthine and ajmalicine, in comparison to cells in suspension. The increased total protein synthesis in immobilized cells suggests a diversion of the primary metabolic flux toward protein biosynthetic pathways and away from other growth processes. In vitro assays for the specific activity of tryptophan decarboxylase (TDC) and tryptophan synthase (TS) suggest that the decreased accumulation of tryptamine in immobilized cells was due to reduced tryptophan biosynthesis. The specific activity of TDC was similar in immobilized and suspension-cultured cells. However, the expression of TS activity in immobilized cells was reduced to less than 25% of the maximum level in suspension-cultured cells. The reduced availability of a free tryptophan pool in immobilized cells is consistent with the reduced TS activity. Reduced tryptamine accumulation, however, was not responsible for the decreased accumulation of indole alkaloids in immobilized cells. Indole alkaloid accumulation increased to a similar level in immobilized and suspension-cultured cells only after the addition of exogenous secolaganin to the culture medium. The addition of tryptophan resulted in increased accumulation of tryptamine, but had no effect on indole alkaloid levels. Reduced biosynthesis of secologanin, the monoterpenoid precursor to indole alkaloids, in immobilized cells is suggested. Immobilization does not appear to alter the activity of indole alkaloid biosynthetic enzymes in our system beyond, and including, strictosidine synthase. Offprint requests to: P. J. Facchini