Cloning and characterization of norbelladine synthase catalyzing the first committed reaction in Amaryllidaceae alkaloid biosynthesis.

BACKGROUND: Amaryllidaceae alkaloids (AAs) are a large group of plant-specialized metabolites displaying an array of biological and pharmacological properties. Previous investigations on AA biosynthesis have revealed that all AAs share a common precursor, norbelladine, presumably synthesized by an enzyme catalyzing a Mannich reaction involving the condensation of tyramine and 3,4-dihydroxybenzaldehyde. Similar reactions have been reported. Specifically, norcoclaurine synthase (NCS) which catalyzes the condensation of dopamine and 4-hydroxyphenylacetaldehyde as the first step in benzylisoquinoline alkaloid biosynthesis. RESULTS: With the availability of wild daffodil (Narcissus pseudonarcissus) database, a transcriptome-mining search was performed for NCS orthologs. A candidate gene sequence was identified and named norbelladine synthase (NBS). NpNBS encodes for a small protein of 19 kDa with an anticipated pI of 5.5. Phylogenetic analysis showed that NpNBS belongs to a unique clade of PR10/Bet v1 proteins and shared 41% amino acid identity to opium poppy NCS1. Expression of NpNBS cDNA in Escherichia coli produced a recombinant enzyme able to condense tyramine and 3,4-DHBA into norbelladine as determined by high-resolution tandem mass spectrometry. CONCLUSIONS: Here, we describe a novel enzyme catalyzing the first committed step of AA biosynthesis, which will facilitate the establishment of metabolic engineering and synthetic biology platforms for the production of AAs.

Exploiting the Catalytic Diversity of Short-Chain Dehydrogenases/Reductases: Versatile Enzymes from Plants with Extended Imine Substrate Scope.

Numerous short-chain dehydrogenases/reductases (SDRs) have found biocatalytic applications in C=O and C=C (enone) reduction. For NADPH-dependent C=N reduction, imine reductases (IREDs) have primarily been investigated for extension of the substrate range. Here, we show that SDRs are also suitable for a broad range of imine reductions. The SDR noroxomaritidine reductase (NR) is involved in Amaryllidaceae alkaloid biosynthesis, serving as an enone reductase. We have characterized NR by using a set of typical imine substrates and established that the enzyme is active with all four tested imine compounds (up to 99 % conversion, up to 92 % ee). Remarkably, NR reduced two keto compounds as well, thus highlighting this enzyme family's versatility. Using NR as a template, we have identified an as yet unexplored SDR from the Amaryllidacea Zephyranthes treatiae with imine-reducing activity (≤95 % ee). Our results encourage the future characterization of SDR family members as a means of discovering new imine-reducing enzymes.

O-acetylserine(thio)lyase (OAS-TL) molecular expression in Pancratium maritimum L. (Amaryllidaceae) under salt stress.

MAIN CONCLUSION: Different levels of salt stress affected the OAS-TL expression levels in Pancratium maritimum organs (bulb, leaf and root). A detailed method has been described for the identification of the conserved domain of the OAS-TL cDNA in sea daffodil given the scarce data available for the Amaryllidaceae family. Pancratium maritimum or sea daffodil (Amaryllidaceae) is a bulbous geophyte growing on coastal sands. In this study, we investigated the involvement of cysteine synthesis for salt tolerance through the expression of the enzyme O-acetylserine(thio)lyase (OAS-TL) during the stress response to NaCl treatments in P. maritimum. Quantitative real-time PCR was used in different organs (bulb, leaf and root).

The Hippeastrum hybridum PepR1 gene (HpPepR1) encodes a functional guanylyl cyclase and is involved in early response to fungal infection.

It is generally known that cyclic GMP widespread in prokaryotic and eukaryotic cells, is involved in essential cellular processes and stress signal transduction. However, in contrast to animals the knowledge about plant guanylyl cyclases (GCs) which catalyze the formation of cGMP from GTP is still quite obscure. Recent studies of plant GCs are focused on identification and functional analysis of a new family of membrane proteins called "moonlighting kinases with GC activity" with guanylyl cyclase catalytic center encapsulated within intracellular kinase domain. Here we report identification and characterization of plasma membrane receptor of peptide signaling molecules - HpPepR1 in Hippeastrum hybridum. Both bioinformatic analysis of amimo acid sequence and in vitro studies revealed that the protein can act as guanylyl cyclase. The predicted amino acid sequence contains highly conserved 14 aa-long search motif in the catalytic center of GCs from lower and higher eukaryotes. Here, we provide experimental evidence to show that the intracellular domain of HpPepR1 can generate cGMP in vitro. Moreover, it was shown that the accumulation of HpPepR1 transcript was sharply increased after Peyronellaea curtisii (=Phoma narcissi) fungal infection, whereas mechanical wounding has no influence on expression profile of studied gene. These results may indicate the participation of cGMP-dependent pathway in rapid, alarm plant reactions induced by pathogen infection.

Transcriptional response of a novel HpCDPK1 kinase gene from Hippeastrum x hybr. to wounding and fungal infection.

Calcium dependent protein kinases (CDPK) are well established plant sensor and effectors for calcium ions and participate in regulation of multiple abiotic and biotic stress responses in plant cells. Here we present the identification and characterization of a new CDPK kinase gene from bulbous plant Hippeastrum x hybr. and examine the role of this kinase in stress responses leading to phytoalexin (PA) production in plant tissues. In the previous research, it was shown that Hippeastrum bulbs mechanically wounded or infected with Peyronellaea curtisii (=Phoma narcissi) are inducted to an antifungal red substance synthesis. In this research, we demonstrated Ca2+ dependence of the phytoalexin production by wounded bulbs. Furthermore, the isolated HpCDPK1 cDNA for ORF was found to be 1596bp long and encoded 531 amino acid protein with CDPK kinase activity, as was shown by recombinant GST-HpCDPK1 enzyme production and analysis. HpCDPK1 transcript was present in all vegetative and chosen generative organs of Hippeastrum plant. The dynamics of the observed HpCDPK1 mRNA changes in bulbs depended on stressor type. The mechanical injury caused one wave of transcript increase while more complex transcript changes were observed within 48h after Peyronellaea inoculation. In plant bulbs already accumulating red phytoalexin, increases in HpCDPK1 mRNA level were observed at certain intervals within 48h whereas, in the case of fungal infection, only one big increment in the transcript amount at the 10th minute after inoculation was detected. The observed transcriptional response of HpCDPK1 gene to wounding and pathogen infection stress suggests a positive correlation with phytoalexin synthesis and maintenance in bulb tissues and puts more light on CDPK kinase role in the plant stress response regulation. This also bears some potential for understanding the mechanism of a phytoalexin formation.

Identification of a Hippeastrum hybridum guanylyl cyclase responsive to wounding and pathogen infection.

Guanosine 3',5'-cyclic monophosphate (cGMP) is a critical component of many (patho)physiological processes in plants whilst guanylyl cyclases (GCs) which catalyse the formation of cGMP from GTP have remained somewhat elusive. Consequently, the two major aims are the discovery of novel guanylyl cyclases and the identification of GC/cGMP mediated processes. To identify a novel GC from Hippeastrum hybridum plant and facilitate the preparation of guanylyl cyclase in an amount sufficient for further crystallographic studies, we have constructed an overproduction system for this enzyme. This gene encodes a protein of 256 amino acids, with a calculated molecular mass of 28kD. The predicted amino acid sequence contains all the typical features and shows a high identity to other plant GCs. The GST-HpGC1 was catalytically active in Escherichia coli cells and the purified, recombinant HpGC1 was able to convert GTP to cGMP in the presence of divalent cations. The used overexpression system yields a guanylyl cyclase as 6% of the bacterial cytosolic protein. Besides the identification of HpGC1 as a guanylyl cyclase, the study has shown that the level of HpCG1 mRNA changed during stress conditions. Both mechanical damage and a Peyronellaea curtisii (=Phoma narcissi) fungi infection led to an initial decrease in the HpGC1 transcript level, followed by a substantial increase during the remainder of the 48-h test cycle. Moreover, significant changes in cyclic GMP level were observed, taking the form of oscillations. In conclusion, our data unequivocally identified the product of the HpGC1 gene as a guanylyl cyclase and demonstrates that such an overproduction system can be successfully used in enzyme synthesis. Furthermore, they indicate a link between the causing stimulus (wounding, infection) and guanylyl cyclase expression and the increase in cGMP amplitude. Therefore, it is concluded that appearance of cyclic GMP as a mediator in defense and wound-healing mechanisms provides a clue to the regulation of these processes.