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Article

Biochemical Characterization of Ornithine Decarboxylases from Solanaceae Plants Producing Tropane Alkaloids

by
Lingjiang Zeng
1,
Tengfei Zhao
1,
Mengxue Wang
1,
Yifan Sun
1,
Chengcun Liu
1,
Xiaozhong Lan
2,
Peng Song
3,* and
Zhihua Liao
1,*
1
Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, SWU-TAAHC Medicinal Plant Joint R&D Centre, School of Life Sciences, Southwest University, Chongqing 400715, China
2
The Provincial and Ministerial Co-Founded Collaborative Innovation Center for R&D in Xizang Characteristic Agricultural and Animal Husbandry Resources, Key Laboratory of Xizang Medicine Resources Conservation and Utilization of Xizang Autonomous Region, TAAHC-SWU Medicinal Plant Joint R&D Center, Xizang Agriculture and Animal Husbandry University, Linzhi 860000, China
3
School of Physical Education, Southwest University, Chongqing 400715, China
*
Authors to whom correspondence should be addressed.
Horticulturae 2025, 11(7), 748; https://doi.org/10.3390/horticulturae11070748
Submission received: 27 May 2025 / Revised: 26 June 2025 / Accepted: 28 June 2025 / Published: 30 June 2025
(This article belongs to the Special Issue Plant Secondary Metabolism and Its Applications in Horticulture)
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Abstract

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in the biosynthesis of polyamines and plant alkaloids, including medicinal tropane alkaloids (TAs). Due to its key role, ODC has been utilized as an effective molecular tool in metabolic engineering. However, to date, only a limited number of plant ODCs have been characterized. Among the reported ODCs, Erythroxylum coca ODC (EcODC) exclusively has ODC activity, while Nicotiana glutinosa ODC (NgODC) exhibits dual ODC and lysine decarboxylase (LDC) activities. The potential LDC activity of ODCs from TA-producing plants remains unknown. Here, we characterized AlODC and DsODC from Anisodus luridus and Datura stramonium, along with two previously reported ODCs from Atropa belladonna (AbODC) and Hyoscyamus niger (HnODC), in Escherichia coli to investigate their enzyme kinetics and substrate specificity. Enzymatic assays revealed that both AlODC and DsODC catalyzed the conversion of ornithine to putrescine, confirming their ODC activity, with AlODC exhibiting a higher catalytic efficiency, comparable to established ODCs. Furthermore, all four ODCs also displayed LDC activity, albeit at significantly lower efficiency (<1% of ODC activity). This study provides a comprehensive analysis of the enzyme kinetics of ODCs from TA-producing plants, identifying promising candidate genes for metabolic engineering for the biomanufacturing of putrescine-derived alkaloids. Moreover, this is the first report of LDC activity in ODCs from Solanaceae TA-producing plants, shedding light on the evolutionary relationship between ODC and LDC.

1. Introduction

Polyamines (PAs) are ubiquitous, low molecular weight nitrogenous aliphatic compounds that are distributed polyamines, distributed in virtually all living systems, including prokaryotes (such as E. coli) [1,2]. Putrescine, spermidine, spermine, and cadaverine are the most common types of PAs [3]. While cadaverine is primarily found in Leguminosae plants and a few other species [4], putrescine, spermine, and spermidine are present in all plants [5]. Studies have shown that polyamines play a crucial role not only in plant growth, development, and stress response, but also in the biosynthesis of plant alkaloids as essential precursors [6,7]. In Leguminosae plants, cadaverine is the original precursor for the biosynthesis of quinolizidine alkaloids (QAs) [7]. Certain QAs exhibit beneficial pharmacological properties, such as cytotoxic, antiarrhythmic, oxytocic, hypoglycemic, and antipyretic effects, making them promising therapeutic agents [8]. In addition to being metabolized into spermine and spermidine in most plants, putrescine can also be converted into nicotine (primarily in tobacco) and medicinal tropane alkaloids (TAs) in plants such as Atropa belladonna, Anisodus luridus, Hyoscyamus niger, and Datura stramonium [9] (Figure 1). As anticholinergic drugs, hyoscyamine is widely used to treat gastrointestinal spasms and bradycardia, while scopolamine is used to prevent motion sickness, control post-operative nausea, and act as adjunct therapy for Parkinson’s disease [10,11]. In Erythroxylum coca, putrescine serves as a crucial precursor in the biosynthesis of cocaine, a substance notorious for being one of the most commonly abused recreational drugs in modern times [12]. Additionally, these polyamines are also important for interaction between bacteria in the rhizosphere and plants [13]. Therefore, cadaverine and putrescine are not only closely related to the growth, development, and stress response of plants but also serve as key precursors in the biosynthesis of plant alkaloids, such as nicotine, QAs, and TAs.
In plants, both putrescine and cadaverine are derived from the decarboxylation of amino acids. Ornithine decarboxylase (ODC) and lysine decarboxylase (LDC) are key enzymes involved in the formation of cadaverine and putrescine by decarboxylating ornithine and lysine, respectively [4] (Figure 1). Putrescine can also be biosynthesized via an additional pathway involving arginine decarboxylase (ADC), as demonstrated in Arabidopsis thaliana [14]. In Leguminosae plants, cadaverine was identified as the primary precursor in the biosynthesis of quinolizidine alkaloids (QAs) [4]. For instance, due to the beneficial pharmacological properties of QAs, LDCs from Leguminosae plants such as Lupinus angustifolius, Sophora flavescens, and Echinosophora koreensis have been cloned and characterized [4]. The overexpression of La-L/ODC in Nicotiana tabacum significantly enhanced the production of cadaverine-derived alkaloids [4]. In A. thaliana, which lacks ODC, heterologous expression of La-L/ODC enhanced polyamine metabolism, phenylpropanoid biosynthesis, and the production of lys-derived alkaloids [15]. In N. tabacum, E. coca, and Solanaceae TA-producing plants, ODC plays a significant role in the biosynthesis of polyamines such as putrescine, spermidine, and spermine, as well as putrescine-derived alkaloids. In N. tabacum, the overexpression of ODC led to enhanced production of polyamines and nicotine, along with improved tolerance to salt stress [16], while the suppression of NtODC inhibited the biosynthesis of polyamines, resulting in delayed flowering, partial male and female sterility [17]. In A. belladonna, the overexpression of AbODC enhanced TA biosynthesis and improved plant tolerance to low temperature [18]. Nowadays, ODC has shown significant potential in improving TA production and plant resistance to abiotic stress.
Due to the important roles of these amino acid decarboxylases in alkaloid biosynthesis and plant growth and development, the enzymatic characteristics of LDC and ODC in certain plants have been identified [4,18]. LDCs had been predominantly studied in Leguminosae plants [4]. On the other hand, ODCs were primarily characterized in Solanaceae plants that produce TAs, such as A. belladonna and H. niger, as well as in nicotine-producing plants like N. glutinosa and cocaine-producing plants like E. coca [19,20,21]. Previous studies have suggested that both LDC and ODC are pyridoxal phosphate (PLP)-dependent enzymes with close evolutionary relationships [4]. Studies on Leguminosae LDCs have demonstrated that they not only facilitate the conversion of lysine to cadaverine but also exhibit ODC activity, thus also named L/ODCs [4]. Furthermore, these LDCs are capable of decarboxylating both lysine and ornithine with similar kinetic properties [4]. In plants, NgODC and EcODC have been tested for their LDC activity [20,21]. NgODC was found to possess both ODC and LDC activities, with LDC activity being much lower than its ODC activity [20]. In contrast, EcODC was found to lack LDC activity [21]. While in TA-producing plants, previous research only investigated the enzymatic characteristics of AbODC and HnODC, focused on their ODC activity [19]; whether the ODCs from Solanaceae TA-producing plants have LDC activity remains uncertain. Therefore, further enzymatic identification of ODC from diverse plant species is crucial for understanding the enzymatic characteristics of plant ODCs and the evolutionary relationship between LDC and ODC.
To provide more alternative molecular tools for tropane alkaloid (TA) metabolic engineering and have a more comprehensive understanding of the enzymatic characterization of ODCs in Solanaceae plants, this study expressed and purified AlODC and DsODC, alongside previously reported AbODC and HnODC. We subsequently determined the enzyme kinetics of AlODC and DsODC for their ODC activities, while the potential LDC activities across all four enzymes were systematically evaluated.

2. Materials and Methods

2.1. Plant Materials

The plants of Atropa belladonna L., Anisodus luridus Link & Otto, Datura stramonium L., and Hyoscyamus niger L. were grown in the plant garden of Southwest University (29°45′ N, 106°30′ E) located in Chongqing, China. Fresh plant materials from roots, stems, and leaves of these plants were collected for gene cloning and tissue expression analysis. After collection, all samples were immediately stored in liquid nitrogen and subsequently harvested for RNA isolation.

2.2. Gene Cloning and Bioinformatics Analysis

Total RNAs were extracted from the roots of A. belladonna, A. luridus, D. stramonium, and H. niger using an RNA plant kit according to the manufacturer’s protocols (Tiangen Biotech, Beijing, China). RNAs were reverse-transcribed into cDNAs for cloning the coding sequences of AbODC, AlODC, DsODC, and HnODC by using a FastKing RT kit (Tiangen Biotech, Beijing, China). The specific primers for cloning the sequences of DsODC, AbODC, and HnODC were designed based on sequences retrieved from the GenBank database. To isolate the coding sequence of AlODC, a pair of gene-specific primers was used based on the sequenced A. luridus transcriptomes [22]. After confirming the accuracy of the cloned sequences through sequencing, multiple alignments were performed using ClustalX 2.0 [23]. A phylogenetic tree was constructed using the neighbor-joining method in MEGA software v.7.0 [24]. Specific primers used are listed in Supplementary Table S1.

2.3. Gene Expression Analysis

To analyze the tissue expression profiles of AbODC, AlODC, DsODC, and HnODC, total RNAs were extracted from the roots, stems, and leaves of A. belladonna, A. luridus, D. stramonium, and H. niger. After reverse transcription into cDNAs, the expression levels of the genes were analyzed by RT-qPCR using the phosphoglycerate kinase gene (PGK) as an internal reference, according to the reported methods [25]. The qPCR kits were purchased from BIO-RAD, and the qPCR system was an IQ5 thermocycler (BIO-RAD, Hercules, CA, USA). The 2−ΔΔCT method was used to calculate the relative gene expression levels [26]. The primers used are listed in Supplementary Table S1.

2.4. Protein Purification and ODC Activity Assays

The coding sequences of AbODC, AlODC, DsODC, and HnODC were individually amplified through PCR using gene-specific primers incorporating BamHI and SacI recognition sequences. These amplified products were subsequently cloned into the pET-28a+ expression vector using the restriction enzymes BamHI and SacI (Figure S2). The recombinant plasmids were transformed into E. coli BL21 (DE3) for heterologous protein production. Transformed bacterial colonies were grown in LB broth supplemented with dual antibiotics (kanamycin 50 μg mL−1 and chloramphenicol 34 μg mL−1) at 37 °C with shaking. Protein expression was initiated by adding 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) when the OD value of bacterial cultures reached 0.6. Post-induction cultivation was conducted at 25 °C for 6 h, and the bacterial cells were subsequently pelleted by centrifugation for subsequent purification processes, according to previously established methods [18]. The recombinant His-tagged ODCs were purified using the ProteinIso® Ni-NTA Resin kit (TransGen, Beijing, China) according to the manufacturer’s protocol and confirmed by SDS-PAGE. Since the kinetic parameters of the ODC activity of AbODC and HnODC have been previously reported in previous studies, showing the highest activity at pH 8.0 and a temperature of 30 °C, and are relatively consistent with the optimal temperature and pH reported for EcODC and NgODC [18], this study also measured the kinetic parameters of AlODC and DsODC for their ODC activity under the same conditions. Each enzymatic assay was performed in 1 mL of enzymatic reaction buffer containing Tris-HCl buffer (pH 8.0), 1 mM DTT, 1 mM PLP, and ornithine (0–8 mM), at a temperature of 30 °C for 60 min. A total of 200 μL of NaOH solution (12 M) was used to terminate the enzymatic reaction. The products in the enzymatic assays and putrescine standards were benzoylated with benzoyl chloride according to methods in a previous study [19]. Then, the benzoylated samples were identified using an Orbitrap Exploris 120 LC-MS (Thermo Scientific, Pittsburgh, PA, USA) and detected according to the methods previously described [18]. The Michaelis–Menten curves of the ODCs were drawn to determine their Km and Vmax values, on which calculations of turnover rate (Kcat) and catalytic efficiency (Kcat/Km) were based. The primers used are listed in Supplementary Table S1.

2.5. LDC Activity Analysis

To investigate whether the ODCs from Solanaceae TA-producing plants have LDC activity, the structures of La-L/ODC (representing L/ODC) and four ODCs (AbODC, AlODC, DsODC, and HnODC) were predicted, and the complex models of enzymes (exemplified by La-L/ODC and AlODC) with both substrates (ornithine and lysine, respectively) were constructed using Proteinix Server according to the reported methods [27]. Subsequently, the LDC activity of purified recombinant AbODC, AlODC, DsODC, and HnODC was tested in this study. Each enzymatic assay was conducted in 1 mL of enzymatic reaction buffer containing Tris-HCl (pH 7.5), 1 mM DTT, 1 mM PLP, and 4 mM lysine. After a 60 min reaction, the reaction was stopped by adding 200 μL of 12 M NaOH. After confirming the presence of their LDC activity, the optimal pH was determined in 40 mM potassium phosphate buffer (pH 6.0–8.0) or 40 mM Tris-HCl buffer (pH 7.2–9.2) containing 1 mM DTT and 1 mM PLP for 60 min using 6 mM lysine as the substrate. To determine the optimal temperature, the purified protein was tested by incubating the reaction mixture in 40 mM potassium phosphate (pH 6.8) for 60 min at different temperatures ranging from 22 to 64 °C. After confirming the optimal temperature and pH, each enzymatic assay was performed in 1 mL of enzymatic reaction buffer containing potassium phosphate buffer (pH 6.8), 1 mM DTT, 1 mM PLP, and lysine (0–8 mM) at a temperature of 52 °C for 60 min. A volume of 200 μL of NaOH solution (12 M) was used to terminate the enzymatic reaction. The products in the enzymatic assays and cadaverine standards were benzoylated into benzoyl cadaverine. Subsequently, the benzoylated samples were identified and detected by referring to the methods for putrescine. The Michaelis–Menten curves of these ODCs were plotted to determine their Km and Vmax values, and calculations of turnover rate (Kcat) and catalytic efficiency (Kcat/Km) were made based on these values.

3. Results

3.1. Gene Cloning and Protein Sequence Alignment

The full-length coding sequence of AlODC is 1293 bp in length and encodes a 430 amino acid polypeptide (Figure S1), which is highly similar to those of AbODC, HnODC, and DsODC. BLASTP analysis indicated a high level of similarity at the amino acid level among ODC proteins in Solanaceae plants, especially in Solanaceae TA-producing plants (Figure 2). The similarities among the ODCs of the four TA-producing plants are almost above 95%, with consistency exceeding 90% (Figure 2). Interestingly, the similarities among plant ODCs and L/ODCs at the amino acid level are higher than those between plant ODCs and animal or microbial ODCs (Figure 2). For example, the similarity between AlODC and La-L/ODC is 67.3%, while the similarity between AlODC and Human ODC (HsODC) is only 47.0% (Figure 2). This suggests that plant ODC and L/ODC may have a closer evolutionary relationship. Motif 1 (YAVKCN) functions as the binding site for the coenzyme pyridoxal-5′-phosphate (PLP) [19], which is present in the amino acid sequences of all ODC and L/ODC (Figure 2). Motif 2 is considered the substrate binding site in ODCs and L/ODCs. In this motif, (Met and Phe) are conserved within L/ODCs from QA-producing plants, while in the eukaryotic ODCs, the amino acid residues (Met and Phe) were changed to (Leu and His) [4] (Figure 2). These results suggest that there may be certain similarities and differences in the catalytic activities of these L/ODCs and ODCs.

3.2. Evolutionary Relationship Analysis

To further explore the evolutionary relationships between the ODC proteins and their relationships with L/ODC proteins, a phylogenetic tree was constructed using MEGA software v.7.0 based on the neighbor-joining (NJ) algorithm (Figure 3). The phylogenetic analysis revealed a close evolutionary relationship among the ODCs of the four Solanaceae plants. Notably, the evolutionary relationship between eukaryotic ODCs and prokaryotic ODCs (e.g., E. coli) was found to be distant, whereas ODCs from Solanaceae plants and L/ODCs from Leguminosae plants clustered within the same branch of the evolutionary tree (Figure 3). This branch is further divided into two distinct subgroups, which were clearly separated from the clade containing human and yeast ODCs (Figure 3). Importantly, the evolutionary distance between Solanaceae plant ODCs and Leguminosae L/ODCs was significantly shorter than that between plant ODCs and human/yeast ODCs (Figure 3). These findings suggest that plant ODCs and Leguminosae L/ODCs likely share a common ancestral origin, but subsequently diverged during evolution to form two distinct enzyme types with specialized functions.

3.3. Gene Expression Analysis

The currently reported genes involved in TA biosynthetic pathways are typically highly expressed in the roots, such as PMT, H6H, and HDH [22]. In order to comprehensively understand the tissue expression patterns of ODC genes in Solanaceae TA-producing plants, we analyzed the tissue expression patterns of AbODC (Figure 4A), AlODC (Figure 4B), DsODC (Figure 4C), and HnODC (Figure 4D) in the organs of roots, stems, and leaves of A. belladonna, A. luridus, D. stramonium, and H. niger using real-time quantitative PCR (RT-qPCR), respectively. The results showed that all the ODC genes of these four species were expressed at a higher level in the roots, where the TAs biosynthesis takes place [28] (Figure 4). In contrast to other pathway genes, like H6H and PMT, ODCs were not only expressed in the roots but also showed certain levels of expression in the leaves and stems, indicating that ODCs may be involved in secondary metabolism in the roots and potentially related to the growth and development of other organs.

3.4. Enzyme Kinetics Analysis of AlODC and DsODC for Their ODC Activities

In this experiment, the recombinant proteins AbODC, AlODC, DsODC, and HnODC were expressed in E. coli using the pET expression system and purified with the ProteinIso® Ni-NTA Resin kit (TransGen Biotech, Beijing, China)). Given that the enzyme kinetics of AbODC and HnODC for their ODC activities had been previously reported [19], the enzyme kinetics of AlODC and DsODC for their ODC activities were analyzed in this study. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the purified proteins were approximately 50 kDa, which was consistent with their predicted sizes (Figure 5A,B). No significant impurity bands were detected, indicating that the purified proteins were of high quality and suitable for subsequent enzymatic assays. The catalyzed products, control samples, and putrescine standard samples were derivatized with benzoyl chloride and analyzed. The results showed that, in the fingerprint chromatograms of the catalytic products of recombinant AbODC and DsODC (Figure 5C), a new absorption peak appeared at a retention time of 10.6 min, which matched the retention time of benzoylated putrescine standard samples. In contrast, no corresponding peak was observed at 10.6 min in the chromatograms of the negative controls. Further confirmation by mass spectrometry analysis demonstrated that the target compounds exhibited the same m/z value (297.25) as that of the benzoylated authentic putrescine samples under positive ESI-MS detection mode (Figure S4). These findings indicated that the purified recombinant AlODC and DsODC possessed the enzymatic activity to catalyze the decarboxylation of ornithine to produce putrescine. Subsequently, the kinetic properties of AlODC and DsODC were investigated at pH 8.0 and 30 °C using ornithine as the substrate (Figure 5D,E). The corresponding enzyme kinetic parameters, derived from the Michaelis–Menten curves, were summarized in Table 1. These results revealed that the ODCs of TA-producing plants generally exhibited higher catalytic efficiency (Kcat/Km) compared to that of NgODC. Among these ODCs, AbODC and AlODC have higher catalytic efficiency and may be better candidate genes for the engineering of putrescine-derived metabolites.

3.5. LDC Activity Identification and Optimal Condition Selection

In plants, EcODC and NgODC have been tested for their LDC activity. While EcODC exclusively demonstrated ODC activity and was unable to catalyze the conversion of lysine to cadaverine, NgODC exhibited both ODC and LDC activities. However, there have been no prior reports on whether ODCs in TA-producing plants possess LDC activity. To validate whether the ODCs from TA-producing plants possess a dual activity similar to L/ODC, we predicted the structures of La-L/ODC (representing L/ODC) and four ODCs (AbODC, AlODC, DsODC, and HnODC) using the Proteinix Server (Figure S3). Structural comparison revealed that La-L/ODC shares significant spatial similarity with these ODCs. Furthermore, the complex models of enzymes (exemplified by La-L/ODC and AlODC) with both substrates (ornithine and lysine, respectively) were constructed using Proteinix Server. The results demonstrate that both La-L/ODC and AlODC exhibit binding pockets compatible with both substrates, indicating both enzymes have the potential to bind and catalyze the decarboxylation of these dual substrates (Figure 6).
In this study, the LDC activities of recombinant proteins AbODC, AlODC, DsODC, and HnODC were investigated (Figure 7). The results revealed a new absorption peak on the fingerprint chromatograms of the catalytic products of these proteins, which corresponded to the retention time of the derivatized cadaverine standard (Figure 7A). No such peak was observed in the blank control at the same retention time (Figure 7A). Further confirmation by mass spectrometry demonstrated that the target compound catalyzed by these recombinant proteins had an m/z value of 311.2 (Figure S4), matching that of the benzoylated cadaverine standard. These findings provide evidence that the purified recombinant proteins, in addition to their ODC activity, also possessed LDC activity. These results indicate that the tested ODCs exhibit dual enzymatic activities, namely ODC and LDC. Combined with the previously reported findings on NgODC, it can be inferred that ODCs in Solanaceae plants commonly possess both ODC and LDC activities. Temperature and pH are crucial factors that influence enzyme activity [29]; thus, studying the optimal temperature and pH is fundamental for enzyme kinetics studies. To investigate the optimal temperature and pH for their LDC activity, in this study, AlODC is presented as an example for elaboration. Results demonstrated that the optimal pH for AlODC catalyzing lysine decarboxylation is near 6.8 (Figure 7B), which was consistent with the optimal pH for NgODC catalyzing lysine decarboxylation [20]. The optimal temperature for AlODC catalyzing lysine decarboxylation is near 52 °C (Figure 7C), which is the first reported optimal temperature for the LDC activity of plant-type ODC proteins. It is worth noting that, at room temperature (22–24 °C), AlODC exhibited a near-complete loss of lysine decarboxylation activity (Figure 7C).

3.6. Enzyme Kinetic Analysis of ODCs for Their LDC Activities

Based on the results mentioned above, enzyme kinetic assays of these ODCs for their LDC activities were conducted under optimal conditions of pH 6.8 and 52 °C. After a 60 min reaction period, the reaction products were derivatized, extracted, and analyzed. Michaelis–Menten kinetic curves were plotted based on the quantified reaction products. The Michaelis–Menten kinetic curves for AbODC (Figure 8A), AlODC (Figure 8B), HnODC (Figure 8C), and DsODC (Figure 8D) are presented in Figure 8. The corresponding enzyme kinetic parameters, derived from the Michaelis–Menten curves, are summarized in Table 1. The results demonstrated that although AbODC, AlODC, DsODC, and HnODC exhibited substrate affinities for lysine, their LDC activities were significantly lower compared to Leguminosae L/ODCs (Table 1). Furthermore, while AbODC, AlODC, DsODC, and HnODC all possessed both ODC and LDC activities, their LDC activities were markedly lower than their ODC activities. These findings suggest that ODCs from Solanaceae plants commonly possess LDC activity in addition to ODC activity and clearly demonstrate the differences and connections in enzymatic characteristics between Solanaceae ODCs and Leguminosae L/ODCs.

4. Discussion

Currently, research on the biological functions of ODC is extensive, but these studies primarily focus on microorganisms and animals [2,13,30,31,32]. Due to its association with cancer, human ODC has garnered significant attention and sparked widespread discussion among scientists [33,34]. In contrast, plant ODCs have not been well investigated. In plants, ODCs are key enzymes involved in the biosynthesis of putrescine, which serves as the rate-limiting step for the production of spermidine, spermine, and putrescine-derived alkaloids such as cocaine, nicotine, and medicinal TAs [35]. Consequently, ODC plays a critical role in plant growth, development, and secondary metabolism. Although DsODC was the first gene cloned from plants, its identification was initially accomplished using only crude protein extracts derived from E. coli expressing DsODC [36]. Over the past years, only NgODC, EcODC, AbODC, and HnODC have been fully characterized with respect to their enzyme kinetics [19], leaving our understanding of plant ODCs relatively limited. Previous studies have demonstrated that ODC played a more significant role in TA biosynthesis compared to ADC and served as an efficient molecular tool for TA metabolic engineering [18,19]. Therefore, investigating ODCs in Solanaceae TA-producing plants holds considerable practical importance.
In this study, ODC genes from A. belladonna, A. luridus, D. stramonium, and H. niger were cloned. Tissue expression analysis revealed that AbODC, AlODC, DsODC, and HnODC were predominantly expressed in the roots, a pattern consistent with the expression of previously identified TA biosynthesis genes, such as H6H and PMT [28]. This suggests that ODC is closely associated with TA biosynthesis in the roots. Unlike H6H and PMT, these ODCs also exhibited some expression in the leaves, indicating that ODCs in Solanaceae plants may not only participate in TA biosynthesis but also play a role in leaf development or other physiological processes. For instance, in tobacco, ODC is involved not only in nicotine biosynthesis but also in overall plant growth and development [37]. In H. niger, suppression of ODC significantly reduced leaf biomass [38], further supporting its role in growth regulation. Amino acid sequence alignment and phylogenetic tree analysis suggested that ODCs in Solanaceae plants might share a common ancestor with Leguminosae L/ODCs. This study found that AbODC, AlODC, DsODC, and HnODC exhibit both ODC and LDC activities, providing robust biochemical evidence for the common ancestry of Solanaceae ODCs and Leguminosae L/ODCs. Interestingly, EcODC from Erythroxylum coca does not exhibit LDC activity [21], a phenomenon that warrants further investigation.
Although these ODCs and Leguminosae L/ODCs possess both ODC and LDC activities, they exhibit significant differences in their kinetic properties. Previous studies have shown that the optimal pH and temperature for ODC activity in Solanaceae plants are typically pH 8.0 and 30 °C [19]. Enzyme kinetic assays revealed that the catalytic efficiency of Solanaceae ODCs for ODC activity is more than 100 times higher than that for LDC activity. In contrast, Leguminosae L/ODCs generally exhibit similar catalytic efficiencies for both lysine and ornithine [4]. Additionally, enzyme studies demonstrated that at room temperature (22–26 °C), AbODC, AlODC, DsODC, and HnODC nearly lack the ability to catalyze the conversion of lysine to cadaverine. These findings suggested that ODCs in Solanaceae TA-producing plants primarily function in putrescine biosynthesis, while their LDC activity may have been retained as an evolutionary relic. Furthermore, it was reported that in Brugmansia candida (syn. Datura candida), a Solanaceae plant producing tropane alkaloids scopolamine and hyoscyamine, endogenous cadaverine could not be detected in the whole plant [39]. Consequently, the combined evidence of low in vitro activity and undetectable product in planta implies that LDC activity in these ODCs is likely a residual property from ancestral decarboxylases. Nevertheless, potential minor roles (e.g., in stress responses or developmental stages) cannot be entirely excluded and warrant targeted future studies. The differences in enzymatic activity between Solanaceae ODCs and Leguminosae L/ODCs align with molecular phylogenetic analyses, indicating that although these enzymes share a common ancestor, they have undergone functional divergence during evolution. Kinetic studies further revealed that AbODC and AlODC exhibit higher catalytic efficiency for ornithine decarboxylation compared to DsODC, HnODC, and other reported plant ODCs, making them superior candidate genes for use as molecular tools in TA metabolic engineering.

5. Conclusions

In summary, this study provides a comprehensive kinetic characterization of ODCs from TA-producing plants. We confirmed the ODC activity of AlODC and DsODC enzymes, with AlODC exhibiting notably high catalytic efficiency comparable to established plant ODCs. Significantly, all four ODCs from Solanaceae TA-producing plants examined LDC activity, albeit at efficiencies below 1% of their ODC activity. This represents the first report of LDC activity in ODCs from Solanaceae TA-producing plants, offering new insights into the functional evolution of these decarboxylases. Our identification of highly efficient ODCs, particularly AlODC, provides valuable genetic tools for metabolic engineering. These enzymes hold significant promise for synthetic biology by efficiently synthesizing putrescine, thereby providing essential precursors for engineered microbes (e.g., engineered yeast or bacteria) to sustainably produceTAs or other putrescine-derived metabolites.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11070748/s1, Table S1: Primers used in qRT-PCR; Figure S1: The coding sequence of AlODC and its deduced amino acid sequence; Figure S2: The procedures to construct the vectors for the prokaryotic expression of the recombinant proteins AbODC, AlODC, DsODC, and HnODC in E. coli; Figure S3: The predicted protein structures of La-L/ODC and ODCs from Solanaceae TA producing plants including AbODC, AlODC, DsODC, and HnODC; Figure S4: MS characterization of benzoylated enzymatic products.

Author Contributions

Conceptualization, L.Z., T.Z. and Z.L.; Methodology, L.Z. and M.W.; Validation, Y.S.; Formal analysis, L.Z. and C.L.; Investigation, L.Z., M.W., Y.S. and C.L.; Resources, X.L. and P.S.; Data curation, Y.S.; Writing—original draft, L.Z.; Writing—review and editing, T.Z., Z.L. and P.S.; Visualization, M.W.; Supervision, T.Z.; Project administration, Z.L.; Funding acquisition, T.Z., X.L. and Z.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Base and Talent Foundation of Science and Technology Department of Xizang Autonomous Region (XZ202501JD0026), the National Natural Science Foundation of China (32300229, 32370277), the National Key Research and Development Program of China (2022YFD1201600), the Forth National Survey of Traditional Chinese Medicine Resources, Chinese or Tibet Medicinal Resources Investigation in Tibet Autonomous Region (State Administration of Chinese Traditional Medicine 20191217–540124 and 20200501–542329).

Data Availability Statement

The data that support the findings of this study are available from the authors upon reasonable request.

Acknowledgments

We extend our gratitude to Fei Qiu for his valuable assistance during Lingjiang Zeng’s LC-MS analytical work.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Horticulturae 11 00748 g001
Figure 1. Representative plant species producing medicinal tropane alkaloids and biosynthetic pathway of tropane alkaloids and quinolizidine alkaloids. ADC, arginine decarboxylase; ODC, ornithine decarboxylase; LDC, lysine decarboxylase.
Figure 1. Representative plant species producing medicinal tropane alkaloids and biosynthetic pathway of tropane alkaloids and quinolizidine alkaloids. ADC, arginine decarboxylase; ODC, ornithine decarboxylase; LDC, lysine decarboxylase.
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Horticulturae 11 00748 g002
Figure 2. Multiple alignments of ornithine decarboxylases and lysine/ornithine decarboxylases. The two motifs are boxed in red. The color gradient indicates different levels of amino acid conservation and sequence similarity. Gene Bank accession numbers are as follows: AbODC, Atropa belladonna (AIC34713); DsODC, Datura stramonium (CAA61121); HnODC, Hyoscyamus niger (QCI44167); NgODC, Nicotiana glutinosa (AAG45222.1); EcODC, Erythroxylum coca (AEQ02350.1); HsODC, Homo sapiens (NP_001274119); ScODC, Saccharomyces cerevisiae (DAA08982.1); La-L/ODC, Lupinus angustifolius (AB560664); Sf-L/ODC, Sophora flavescens (AB561138); Ek-L/ODC, Echinosophora koreensis (AB561139).
Figure 2. Multiple alignments of ornithine decarboxylases and lysine/ornithine decarboxylases. The two motifs are boxed in red. The color gradient indicates different levels of amino acid conservation and sequence similarity. Gene Bank accession numbers are as follows: AbODC, Atropa belladonna (AIC34713); DsODC, Datura stramonium (CAA61121); HnODC, Hyoscyamus niger (QCI44167); NgODC, Nicotiana glutinosa (AAG45222.1); EcODC, Erythroxylum coca (AEQ02350.1); HsODC, Homo sapiens (NP_001274119); ScODC, Saccharomyces cerevisiae (DAA08982.1); La-L/ODC, Lupinus angustifolius (AB560664); Sf-L/ODC, Sophora flavescens (AB561138); Ek-L/ODC, Echinosophora koreensis (AB561139).
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Horticulturae 11 00748 g003
Figure 3. Phylogenetic analysis of ODC and L/ODC enzymes. The phylogenetic tree was constructed using the neighbor-joining method. The numbers on the branches indicated the boot-strapped values. The scale represented the genetic distance. Gene Bank accession numbers are as follows: Atropa belladonna (AIC34713), Datura stramonium (CAA61121), Hyoscyamus niger (QCI44167), Nicotiana glutinosa (AAG45222.1), Erythroxylum coca (AEQ02350.1), Homo sapiens (NP_001274119), Saccharomyces cerevisiae (DAA08982.1), Lupinus angustifolius (AB560664), Sophora flavescens (AB561138), Echinosophora koreensis (AB561139), Escherichia coli (BAE77028.1). Horticulturae 11 00748 i001 represents those reported ODCs. Horticulturae 11 00748 i002 represents those reported L/ODCs.
Figure 3. Phylogenetic analysis of ODC and L/ODC enzymes. The phylogenetic tree was constructed using the neighbor-joining method. The numbers on the branches indicated the boot-strapped values. The scale represented the genetic distance. Gene Bank accession numbers are as follows: Atropa belladonna (AIC34713), Datura stramonium (CAA61121), Hyoscyamus niger (QCI44167), Nicotiana glutinosa (AAG45222.1), Erythroxylum coca (AEQ02350.1), Homo sapiens (NP_001274119), Saccharomyces cerevisiae (DAA08982.1), Lupinus angustifolius (AB560664), Sophora flavescens (AB561138), Echinosophora koreensis (AB561139), Escherichia coli (BAE77028.1). Horticulturae 11 00748 i001 represents those reported ODCs. Horticulturae 11 00748 i002 represents those reported L/ODCs.
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Horticulturae 11 00748 g004
Figure 4. Expression analysis of ODCs. Expression of AbODC (A), AlODC (B), DsODC (C), and HnODC (D) in leaf, stem, and root was determined by RT-qPCR. Different lowercase letters indicate significant differences (p < 0.05). Vertical bars represent means ± standard errors (n ≥ 3).
Figure 4. Expression analysis of ODCs. Expression of AbODC (A), AlODC (B), DsODC (C), and HnODC (D) in leaf, stem, and root was determined by RT-qPCR. Different lowercase letters indicate significant differences (p < 0.05). Vertical bars represent means ± standard errors (n ≥ 3).
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Horticulturae 11 00748 g005
Figure 5. Purification and enzymatic assays of recombinant His-tagged AlODC and DsODC for their ODC activities. (A) Purification of recombinant His-tagged AlODC. (B) Purification of recombinant His-tagged DsODC. (C) HPLC traces of ODC-catalyzed products, putrescine standards, and negative control samples. (D) The Michaelis–Menten curve of AlODC for its ODC activity. (E) The Michaelis–Menten curve of DsODC for its ODC activity. The red arrow represents the the benzoylated putrescine. M, protein maker; AlODC, recombinant protein of His tagged-AlODC; DsODC, recombinant protein of His tagged-AlODC; Error bars in D and E indicate SD (n = 3).
Figure 5. Purification and enzymatic assays of recombinant His-tagged AlODC and DsODC for their ODC activities. (A) Purification of recombinant His-tagged AlODC. (B) Purification of recombinant His-tagged DsODC. (C) HPLC traces of ODC-catalyzed products, putrescine standards, and negative control samples. (D) The Michaelis–Menten curve of AlODC for its ODC activity. (E) The Michaelis–Menten curve of DsODC for its ODC activity. The red arrow represents the the benzoylated putrescine. M, protein maker; AlODC, recombinant protein of His tagged-AlODC; DsODC, recombinant protein of His tagged-AlODC; Error bars in D and E indicate SD (n = 3).
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Horticulturae 11 00748 g006
Figure 6. The complex model of a small molecule with predicted protein structures of L/ODC and ODC. (A) The complex model of lysine and PLP with predicted protein structures of La-L/ODC. (B) The complex model of ornithine and PLP with predicted protein structures of La-L/ODC. (C) The complex model of lysine and PLP with predicted protein structures of AlODC. (D) The complex model of ornithine and PLP with predicted protein structures of AlODC.
Figure 6. The complex model of a small molecule with predicted protein structures of L/ODC and ODC. (A) The complex model of lysine and PLP with predicted protein structures of La-L/ODC. (B) The complex model of ornithine and PLP with predicted protein structures of La-L/ODC. (C) The complex model of lysine and PLP with predicted protein structures of AlODC. (D) The complex model of ornithine and PLP with predicted protein structures of AlODC.
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Figure 7. LDC activity identification and optimal condition selection. (A) HPLC traces of ODC-catalyzed products, cadaverine standards, and negative control samples. (B) LDC activity under different pH conditions. (C) LDC activity at different temperatures. Error bars in C and D indicate SD (n = 3).
Figure 7. LDC activity identification and optimal condition selection. (A) HPLC traces of ODC-catalyzed products, cadaverine standards, and negative control samples. (B) LDC activity under different pH conditions. (C) LDC activity at different temperatures. Error bars in C and D indicate SD (n = 3).
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Horticulturae 11 00748 g008
Figure 8. Enzyme kinetic analysis of ODCs for their LDC activities. (A) The Michaelis–Menten curve of AbODC for its LDC activity. (B) The Michaelis–Menten curve of AlODC for its LDC activity. (C) The Michaelis–Menten curve of HnODC for its LDC activity. (D) The Michaelis–Menten curve of DsODC for its LDC activity. Error bars indicate SD (n = 3).
Figure 8. Enzyme kinetic analysis of ODCs for their LDC activities. (A) The Michaelis–Menten curve of AbODC for its LDC activity. (B) The Michaelis–Menten curve of AlODC for its LDC activity. (C) The Michaelis–Menten curve of HnODC for its LDC activity. (D) The Michaelis–Menten curve of DsODC for its LDC activity. Error bars indicate SD (n = 3).
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Table 1. Kinetic parameters of L/ODCs and ODCs.
Table 1. Kinetic parameters of L/ODCs and ODCs.
Km (mM)Vmax
(nmol min−1 μg−1)		Kcat (s−1)Kcat/Km (M−1s−1)Ratio of Kcat/KmReference
ProteinODCLDCODCLDCODCLDCODCLDCODC/LDC
AlODC1.191.301.095.47 × 10−30.914.57 × 10−37613.52216this study
DsODC3.111.131.945.54 × 10−31.624.64 × 10−35234.11127this study
AbODC1.031.900.937.69 × 10−30.676.42 × 10−36493.38192this study and [19]
HnODC2.621.591.878.49 × 10−31.577.10 × 10−35994.47134this study and [19]
EcODC0.40ND0.25ND0.18ND465ND-[21]
NgODC0.561.591.25 × 10−21.52 × 10−49.3 × 10−31.13 × 10−416.530.07233[20]
La-L/ODC1.052.371.141.490.911.188594331.90[4]
Sf-L/ODC1.532.101.462.941.162.2375511080.68
Ek-L/ODC1.323.850.762.420.731.914544690.96
Footer: Kinetic parameters of AbODC and HnODC for their ODC activity were obtained or recalculated from previously reported studies [19], while kinetic parameters for their LDC activity were analyzed in this study. Kinetic parameters of EcODC, NgODC, and three L/ODCs listed in Table 1 were obtained or recalculated from published data [4,20,21].
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Zeng, L.; Zhao, T.; Wang, M.; Sun, Y.; Liu, C.; Lan, X.; Song, P.; Liao, Z. Biochemical Characterization of Ornithine Decarboxylases from Solanaceae Plants Producing Tropane Alkaloids. Horticulturae 2025, 11, 748. https://doi.org/10.3390/horticulturae11070748

AMA Style

Zeng L, Zhao T, Wang M, Sun Y, Liu C, Lan X, Song P, Liao Z. Biochemical Characterization of Ornithine Decarboxylases from Solanaceae Plants Producing Tropane Alkaloids. Horticulturae. 2025; 11(7):748. https://doi.org/10.3390/horticulturae11070748

Chicago/Turabian Style

Zeng, Lingjiang, Tengfei Zhao, Mengxue Wang, Yifan Sun, Chengcun Liu, Xiaozhong Lan, Peng Song, and Zhihua Liao. 2025. "Biochemical Characterization of Ornithine Decarboxylases from Solanaceae Plants Producing Tropane Alkaloids" Horticulturae 11, no. 7: 748. https://doi.org/10.3390/horticulturae11070748

APA Style

Zeng, L., Zhao, T., Wang, M., Sun, Y., Liu, C., Lan, X., Song, P., & Liao, Z. (2025). Biochemical Characterization of Ornithine Decarboxylases from Solanaceae Plants Producing Tropane Alkaloids. Horticulturae, 11(7), 748. https://doi.org/10.3390/horticulturae11070748

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