Next Article in Journal
Cytokine Networks in Lichen Sclerosus: A Roadmap for Diagnosis and Treatment?
Previous Article in Journal
Amine-Functionalized Mesoporous Silica for Efficient CO2 Capture: Stability, Performance, and Industrial Feasibility
Previous Article in Special Issue
Overexpression of the Glycyrrhiza uralensis Phenylalanine Ammonia-Lyase Gene GuPAL1 Promotes Flavonoid Accumulation in Arabidopsis thaliana
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 26
Issue 9
10.3390/ijms26094314
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Genome-Wide Identification and Functional Analysis of the Norcoclaurine Synthase Gene Family in Aristolochia contorta

by
Yayun Xu
1,2,
Sixuan Zhang
1,2,
Fanqi Meng
1,2,
Wenjing Liang
1,2,
Yunliang Peng
1,2,
Butuo Zhu
1,2,
Lili Niu
1,2,
Chunling Wang
1,2,
Caili Li
1,2,* and
Shanfa Lu
1,2,*
1
State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-Di Herbs, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China
2
Key Lab of Chinese Medicine Resources Conservation, State Administration of Traditional Chinese Medicine of the People’s Republic of China, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2025, 26(9), 4314; https://doi.org/10.3390/ijms26094314
Submission received: 21 March 2025 / Revised: 24 April 2025 / Accepted: 29 April 2025 / Published: 1 May 2025
(This article belongs to the Special Issue Biosynthesis and Regulatory Mechanism of Secondary Metabolites in Medicinal Plants: 2nd Edition)
Browse Figures
Versions Notes

Abstract

Aristolochia contorta Bunge has been widely used as traditional Chinese medicine materials. However, its utility faces a great challenge due to the presence of aristolochic acids (AAs), a class of benzylisoquinoline alkaloid (BIA) derivatives. The first step in BIA skeleton formation is catalysis by norcoclaurine synthase (NCS). To gain knowledge of BIA and AA biosynthesis in A. contorta, genome-wide characterizations of NCS genes were carried out. This resulted in the identification of 15 A. contorta NCSs, namely, AcNCS1–AcNCS15. The AcNCS1–AcNCS8 proteins contained one catalytic domain, whereas the AcNCS9–AcNCS15 proteins had two. Phylogenetic analysis shows that AcNCS proteins can be classified into two clades. Gene expression analysis shows that five AcNCSs, including AcNCS2, AcNCS4, AcNCS5, AcNCS14, and AcNCS15, exhibited relatively high expression in roots and flowers, where norcoclaurine accumulated. An enzyme catalytic activity assay shows that all five of the AcNCSs can catalyze norcoclaurine formation with AcNCS14 and AcNCS15, exhibiting higher catalytic efficiency. Precolumn derivatization analysis shows that the formed norcoclaurine included (S)- and (R)-norcoclaurine, with more (S)-configuration. The results provide useful information for further understanding BIA and AA biosynthesis in A. contorta and for AA elimination and bioactive compound improvement in AA-containing medicinal materials.

1. Introduction

Aristolochia contorta Bunge belongs to the Aristolochia genus of the plant family Aristolochiaceae Juss. in Piperales. It is distributed in China, Korea, Russia, and Japan and has been widely used as traditional Chinese medicine (TCM) materials. A. contorta has anti-inflammatory, antibacterial, anti-tumor, and other pharmacological activities, and it has been used to treat various diseases, such as inflammation, asthma, and bacterial infection [1,2,3]. It is rich in bioactive compounds, such as alkaloids, terpenoids, and flavonoids [4,5,6]. Among them, alkaloids, such as magnoflorine, reticuline, glaziovine, norcorydine, isocorydine, oxonuciferine, and demethylsonodione, are an important class of bioactive compounds in A. contorta [7,8,9,10,11,12]. These alkaloids belong to benzylisoquinoline alkaloids (BIAs) and their derivatives. A. contorta is also rich in aristolochic acids (AAs), a class of BIA derivatives. They were listed as category 1 carcinogens by the International Agency for Research on Cancer (IARC) in 2008, since they can cause nephrotoxicity and carcinogenicity through induction of gene mutations in animal cells [13,14,15,16,17,18]. Therefore, the use of A. contorta as medicinal materials is met with great challenges and officially forbidden in China.
The elimination of AAs and increase in other bioactive compounds have become a focus of research on this plant species [11,12,19]. In order to achieve this goal, the whole genome of A. contorta was recently sequenced [11]. The catalytic functions of two A. contorta O-methyltransferases, including AcOMT1 and AcOMT2, and two cytochrome P450 proteins CYPs, including AcCYP80G7 and AcCYPQ8, were elucidated. All of them are involved in BIA biosynthesis-related pathways. AcOMT1 and AcOMT2 act as norcoclaurine 6-O methyltransferase to catalyze the conversion of norcoclaurine to coclaurine [11]. AcCYP80G7 catalyzes the conversion of reticuline to hexacyclic aporphine corytuberine [12]. AcCYP80Q8 act as a glaziovine synthase (GS) to catalyze the conversion of (S)- and (R)-N-methylcoclaurine to proaporphine alkaloid (6aS)- and (6aR)-glaziovine [12]. In addition to (S)- and (R)-N-methylcoclaurine, AcCYP80Q8 also accepts (S)- and (R)-coclaurine as substrates for the production of proaporphine alkaloid (S)- and (R)-crotsparine [12].
Norcoclaurine synthase (NCS; EC 4.2.1.78), a Pictet–Spenglerase, catalyzes the condensation of dopamine- and tyrosine-derived 4-hydroxyphenylacetaldehyde (4-HPAA) to form norcoclaurine, the central precursor of BIAs [20]. The reaction generates the isoquinoline core structure and is the first common committed step in the BIA biosynthesis pathway [21]. NCS belongs to the pathogen-related 10/Bet v (PR10/Bet v) protein superfamily [22,23]. It has a catalytic function and plays a crucial role in regulating the biosynthesis of BIAs and derivatives due to its position at the entry point of the BIA pathway [22,24]. The first NCS gene was isolated from Thalictrum flavum (TfNCS) based on empirical amino acid sequences of peptides [22,25,26]. Sequence comparison of TfNCS and Papaver somniferum ESTs resulted in the identification of two opium poppy NCS isoforms, namely, PsNCS1 and PsNCS2 [27]. Subsequently, CjNCS1 was identified by screening Coptis japonica ESTs, and CjPR10A was isolated based on sequence homology with TfNCS [28]. So far, NCS genes have been identified from various plant species, such as Argemone mexicana, Nelumbo nucifera, P. bracteatum, Phellodendron amurense, Sinopodophyllum hexandrum, and Stephania tetrandra [24,29,30,31,32,33]. The results suggest that NCS genes belong to a multigene family. For instance, there are 11 members in C. chinensis, five in Aquilegia coerulea, and three in Macleaya cordata [34]. They can be classified into two clades, including NCS I and NCS II [31]. Among them, members of the NCS II clade are widely distributed across plants, such as Citrus sinensis, Medicago truncatula, Prunus persica, and Cucumis sativus. The distribution of NCS I clade members is limited to plant species producing BIAs, such as P. somniferum, A. mexicana, Corydalis saxicola, Eschscholzia californica, C. japonica, T. flavum, and S. hexandrum [31].
Chirality is a critical factor in the pharmacological activity of drugs. Different enantiomers often exhibit distinct biological effects [35]. For example, (−)-morphine, a well-known BIA, is a potent analgesic, whereas its enantiomer, (+)-morphine, is pharmacologically inactive [36]. This enantioselectivity underscores the importance of understanding the enzymatic mechanisms that control stereochemistry. In the BIA biosynthesis pathways, two key enzymes are responsible for the differential transformation of stereochemistry. The first is NCS, which synthesizes the core structure of BIAs. The stereochemistry of its product establishes the chiral foundation for subsequent BIA compounds [22]. The second is STORR [(S)- to (R)-reticuline], also known as reticuline epimerase (REPI) [37,38,39,40,41]. It catalyzes the stereochemical inversion of (S)-reticuline to (R)-reticuline and plays a crucial role in modulating the stereochemistry in the biosynthesis of morphinan alkaloids [37,38]. STORR is a unique protein fusion that contains a cytochrome P450 domain and an aldo–keto reductase domain [37]. The cytochrome P450 domain is responsible for the oxidation of (S)-reticuline to 1,2-dehydroreticuline, which is subsequently reduced to (R)-reticuline by the aldo–keto reductase domain [37]. The interplay among these enzymes ensures the production of enantiomerically pure BIAs, which are essential for their pharmacological efficacy. The currently reported NCS enzymes are predominantly found in Ranunculales plants, where they stereoselectively generate (S)-norcoclaurine [24,25,27,42,43]. However, NCS enzymes from N. nucifera can simultaneously produce both (S)- and (R)-norcoclaurine [44]. In addition, an NCS from the gymnosperm Gnetum montanum generates the majority of the products as (S)-norcoclaurine and a minor amount as (R)-norcoclaurine [44].
Although NCS genes have been identified in various plant species, there is no information on NCSs from magnoliids, including the medicinal plant A. contorta, which produces bioactive BIAs and carcinogenic AAs. The chirality of BIAs in A. contorta is also unknown. With the long-term goals of elucidating the biosynthesis pathway of BIAs and their derivatives in A. contorta, manipulating the accumulation of BIAs, removing AAs from AA plants, and producing A. contorta BIAs through synthetic biology approaches, the genome-wide identification and subsequent analysis of A. contorta NCS genes (AcNCSs) was carried out. In this study, a total of 15 AcNCS genes were identified and characterized. Phylogenetic analysis shows that NCSs can be classified into three clades. Among them, AcNCS proteins were included in two clades. Gene expression analysis shows that AcNCS2, AcNCS4, AcNCS5, AcNCS14, and AcNCS15 are expressed at relatively high levels in norcoclaurine-accumulated roots and flowers. Molecular cloning and functional characterization show that all five of the AcNCS proteins analyzed can catalyze the condensation of dopamine and 4-HPAA to form (S)- and (R)-norcoclaurine. Among them, AcNCS14 and AcNCS15 exhibit high activity toward dopamine and 4-HPAA.

2. Results

2.1. Genome-Wide Identification of 15 AcNCS Genes in A. contorta

In order to identify AcNCS genes in A. contorta, a tBLASTp analysis of TfNCS, PsNCS1, PsNCS2, and CjNCS2/CjPR10A proteins against the whole genome assembly of A. contorta was carried out [11,25,26,27,28]. Subsequent gene prediction of the retrieved genomic DNA sequences identified a total of 15 full-length members of the AcNCS gene family. They were designated as AcNCS1–AcNCS15, respectively (Table 1 and Table S1). Among them, AcNCS6 and AcNCS7 share 100% identity at the nucleotide and amino acid levels. Chromosome localization analysis shows that 15 AcNCS genes are distributed on three of the seven A. contorta chromosomes, including LG01, LG02, and LG03 (Figure 1a and Table 1) [11]. Among them, LG01 contains two AcNCSs, including AcNCS8 and AcNCS13, which are clustered together. LG03 contains one AcNCS, namely, AcNCS3. LG02 contains the other twelve AcNCSs that are clustered at three sites, with AcNCS4–AcNCS7 at one site; AcNCS1, AcNCS9–AcNCS11, and AcNCS14 at the second site; and AcNCS2, AcNCS12, and AcNCS15 at the third site (Figure 1a). Gene structure analysis shows that 10 of the 15 AcNCSs, including AcNCS1–AcNCS8, AcNCS11, and AcNCS12 have an intron, whereas the other 5 have two introns (Figure 1b and Table 1). The deduced AcNCS proteins range from 160 to 355 amino acids in length (Table 1). Their molecular weights range from 17.6 to 39.0 kDa, and the isoelectric points range from 4.63 to 6.15 (Table 1).

2.2. Phylogenetic Relationships of AcNCS Proteins

In order to analyze the relationship between AcNCSs and NCSs from other plant species, as well as the classification of plant NCSs, a phylogenetic tree was constructed for 15 AcNCSs, 40 NCSs from other plants, and 11 PR10 proteins (Figure 2). The results show that these proteins can be divided into four clades, including three NCS clades and a PR10 clade. Clades NCS I and NCS II have previously been reported [31], whereas NCS III is a novel clade. AcNCSs were included in two of the three clades. Among them, five AcNCSs, including AcNCS3–AcNCS7, were included in clade NCS II, which has members that are widely distributed across plants. The other ten AcNCSs were included in the novel NCS III clade. No AcNCSs were found in clade NCS I, which has members from Ranunculales [31]. This indicates an evolutionary divergence between AcNCSs and Ranunculales NCSs.

2.3. Conserved Domain and Motifs of AcNCS Proteins

NCSs belong to the Bet v 1 protein family. The catalytic domain of NCS proteins, known as the Bet v 1 domain, is highly conserved in plants. In order to determine whether the identified AcNCSs contain the Bet v 1 domain, a conserved domain analysis was performed. The results show that all 15 AcNCS proteins contain the Bet v l domain (Figure 3a). Among them, AcNCS1–AcNCS8 have one Bet v 1 domain, whereas AcNCS9–AcNCS15 contain two. In addition to the conserved domains, conserved motifs could be important for the function of NCSs. To gain preliminary knowledge of the identified AcNCSs, conserved motifs were analyzed using MEME (https://meme-suite.org/meme/ accessed on 25 October 2024), with an e-value cutoff of 1 × 10−10 applied for recognition. This allowed us to identify a total of 10 conserved motifs (motifs 1–10) (Figure 3b and Table S4). The number of motifs in each NCS varied from four to nine. Among the 10 motifs, motifs 1, 2, 4, and 6, which are located in the Bet v 1 domain, were conserved in all of the NCSs analyzed. Motif 9 was specific to AcNCS14 and AcNCS15. Motif 10 was found in AcNCS4, AcNCS5, AcNCS6, AcNCS7, and NnACS1. Multiple amino acid sequence alignments of AcNCSs, TfNCS, and NnNCS1 show that all contain a P-loop conserved in PR10/Bet v 1 family proteins [22]. In addition, previous studies identified four highly conserved residues, including Tyr, Glu, Lys, and Asp, in TfNCS [46]. A sequence comparison of AcNCSs, TfNCS, and NnNCS1 shows that the Glu and Lys residues were more conserved, whereas Tyr and Asp were less conserved (Figure 4). These results indicate the conservation and diversity of AcNCSs.

2.4. Differential Expression of AcNCS Genes in A. contorta

Gene expression patterns are usually associated with gene functions. In order to preliminarily analyze the in vivo roles of AcNCS genes, their expression patterns in roots, stems, leaves, and flowers of A. contorta were detected using quantitative real-time PCR (qRT-PCR). The results show that AcNCS genes were differentially expressed (Figure 5). For instance, AcNCS1 was mainly expressed in stems, followed by leaves. AcNCS2 and AcNCS10–AcNCS13 were predominantly expressed in roots. AcNCS3 was predominantly expressed in roots and flowers. AcNCS4 exhibited relatively high expression in flowers, followed by roots. AcNCS5 was predominantly expressed in flowers. AcNCS6 and AcNCS7 were mainly expressed in stems, leaves, and flowers. AcNCS9 was mainly expressed in leaves, followed by stems and flowers. AcNCS14 and AcNCS15 were mainly expressed in roots, followed by flowers (Figure 5).
Further analysis of the gene expression using the RNA-seq data that we obtained previously shows that the AcNCSs expressed in roots mainly included AcNCS1–AcNCS5, AcNCS10, AcNCS13, AcNCS14, and AcNCS15. Those expressed in flowers mainly included AcNCS4, AcNCS5, AcNCS14, and AcNCS15 (Figure 6a) [11]. The patterns from the RNA-seq analysis were similar to those obtained from qRT-PCR, although slight discrepancies were observed (Figure 6a). This suggests consistency between the qRT-PCR detection and RNA-seq data analysis.

2.5. Accumulation of Norcoclaurine in Roots and Flowers of A. contorta

NCSs catalyze the formation of norcoclaurine, the central precursor of BIAs [20]. In order to investigate the relationship between the expression of AcNCSs and the accumulation of norcoclaurine, we determined the contents of norcoclaurine in roots, stems, leaves, and flowers using LC-MS/MS. The results show that norcoclaurine mainly accumulates in the flowers and roots of A. contorta (Figure 6b). Its contents in roots, stems, leaves, and flowers were 24.61, 7.20, 6.03, and 62.56 μg g−1 DW, respectively. Flowers were the most abundant in norcoclaurine, followed by roots. The contents of norcoclaurine in stems and leaves were relatively low (Figure 6b). Based on the expression of AcNCSs and the accumulation of norcoclaurine, we speculate that AcNCS2, AcNCS4, AcNCS5, AcNCS14, and AcNCS15 could be the main AcNCS proteins responsible for norcoclaurine production in A. contorta.

2.6. AcNCS Proteins Catalyze the Formation of Norcoclaurine

In order to further investigate the catalytic activity of the identified AcNCSs, five AcNCSs, including AcNCS2, AcNCS4, AcNCS5, AcNCS14, and AcNCS15, were selected based on the patterns of gene expression and norcoclaurine accumulation. The coding sequences of the AcNCS genes were cloned by PCR amplification and then ligated into the pET-30a vector. Expression of the AcNCS proteins was carried out using an E. coli expression system. After induction and purification, an in vitro catalytic activity assay of the recombinant AcNCS proteins was assessed using dopamine and 4-HPAA as substrates (Figure 7a). Δ19TfNCS from T. flavum and NnNCS1 from N. nucifera were used as positive controls. UPLC analysis shows that new peaks at a retention time of 5.4 min were generated under the catalysis of AcNCS2, AcNCS4, AcNCS5, AcNCS14, AcNCS15, Δ19TfNCS, and NnNCS1 recombinant proteins (Figure 7b). Their retention time is the same as that of the reference standard (S,R)-norcoclaurine (Figure 7b). Mass spectrum analysis shows that the new products and (S,R)-norcoclaurine had m/z values of 272 (Figure 7c). MS/MS spectrum analysis shows that the new products with m/z values of 272 were fragmented into main ions identical to those of the reference standard (S,R)-norcoclaurine (Figure 7c). The results suggest that the AcNCS proteins analyzed could catalyze the condensation of dopamine and 4-HPAA to norcoclaurine.

2.7. Affinity of AcNCSs Toward Dopamine and 4-HPPA

In order to characterize the detailed kinetics of AcNCS catalysis and investigate the differences in catalytic efficiency among the five AcNCSs, the affinities of the AcNCSs toward dopamine and 4-HPPA were analyzed. To test the affinities of the AcNCSs toward dopamine, AcNCS proteins were incubated with different concentrations of dopamine while keeping 4-HPPA saturate. The results show that the kinetic parameters of the different recombinant AcNCS proteins varied (Table 2). The Km values varied from 420.8 µM for AcNCS14 to 534.9 µM for AcNCS2. The Vmax values varied from 1.6 µM min−1 for AcNCS2 to 2.3 µM min−1 for AcNCS14 and 2.2 µM min−1 for AcNCS15. The Kcat/Km values varied from 3.9 × 104 M−1 S−1 for AcNCS2 to 4.3 × 104 M−1 S−1 for AcNCS15 and 5.3 × 104 M−1 S−1 for AcNCS14 (Table 2). This suggests that AcNCS14 and AcNCS15 have relatively high affinities toward dopamine, whereas the affinities of AcNCS2, AcNCS4, and AcNCS5 toward dopamine are relatively low.
To assess the affinities of the AcNCSs toward 4-HPPA, dopamine was kept saturate, whereas the concentration levels of 4-HPPA varied. The results show that the Km values of the AcNCSs for 4-HPPA varied from 495.3 to 536.4 µM. The Vmax values varied from 1.30 to 1.5 µM min−1. The Kcat/Km values varied from 2.5 × 104 to 2.9 × 104 M−1 S−1 (Table 3). This suggests that AcNCS14 and AcNCS15 have relatively high affinities toward 4-HPPA, whereas the affinities of the other AcNCSs toward 4-HPPA are relatively low. The results are similar to the affinities of the AcNCSs toward dopamine (Table 2).

2.8. Existence of (S)- and (R)-Norcoclaurine in AcNCS-Catalyzed Products

Chirality could significantly affect the pharmacological activity of BIA alkaloids. NCSs are the first enzymes determining the chirality of BIAs. Previous studies show that NCSs from Ranunculales plants catalyze the generation of (S)-norcoclaurine, whereas those from N. nucifera catalyzed the production of both (S)- and (R)-norcoclaurine [24,25,27,42,43,44]. In addition, the reaction catalyzed by a gymnosperm G. montanum NCS generated the majority of products as (S)-norcoclaurine and a minor amount as (R)-norcoclaurine [44]. In order to elucidate the stereochemical characteristics of AcNCS products, we implemented a precolumn derivatization strategy through the application of R-(−)-DBD-PyNCS, as previously described [44]. NnNCS1 from N. nucifera and △19TfNCS from T. flavum were synthesized and used as positive controls. The results show that TfNCS specifically catalyzed the production of (S)-norcoclaurine, whereas NnNCS1 catalyzed the generation of an almost equal amount of (S)- and (R)-norcoclaurine (Figure 8), which is consistent with previous studies [44]. All of the AcNCS-catalyzed products exhibited two distinct enantiomers, with (S)-norcoclaurine being more abundant and (R)-norcoclaurine being less (Figure 8). The difference between the amounts of (S)- and (R)-norcoclaurine varied among the AcNCSs. The products generated under the catalysis of AcNCS14 showed the most significant difference (Figure 8). The results suggest that all five AcNCSs, including AcNCS2, AcNCS4, AcNCS5, AcNCS14, and AcNCS15, catalyze the production of (S)- and (R)-norcoclaurine, with (S)-norcoclaurine being more abundant than (R)-norcoclaurine.

3. Discussion

NCSs are important enzymes catalyzing the first committed step in the biosynthesis of BIAs and their derivatives in plants [21]. Since the first report of an NCS gene from T. flavum, various NCS genes have been identified and characterized from the order Ranunculales [22,25,26,27,28]. In addition, five NCSs from N. nucifera that belong to the order Proteales and an NCS from the gymnosperm G. montanum were also functionally analyzed [44]. Both Ranunculales and Proteales are positioned at the basal nodes of eudicots, whereas A. contorta belongs to a relatively basal node of angiosperms, although the taxonomic status of Magnoliids that A. contorta belongs to has long been debated [11,24,25,27,42,43,47]. From an evolutionary perspective, the identification and characterization of the 15 AcNCS genes is significant for elucidating the evolution history of plant NCS genes.
The phylogenetic analysis shows that AcNCS1, AcNCS2, and AcNCS8–AcNCS15 were included in clade NCS III (distinct from Ranunculales NCSs), whereas AcNCS3–AcNCS7 were included in clade NCS II (Figure 2). Catalytic activity analysis shows that AcNCS2, AcNCS14, and AcNCS15 from clade NCS III and AcNCS4 and AcNCS5 from clade NCS II could catalyze the condensation of dopamine and 4-HPPA to form norcoclaurine (Figure 7a). Taken together with previous results, this shows that NnNCS1, NnNCS3, NnNCS5, and NnNCS7 from clade NCS I and NnNCS4 from clade NCS II could catalyze the reaction [44]; it is possible that members of all three NCS clades have the potential to catalyze norcoclaurine formation. Considering that the catalytic activity and substrate affinities of the NCSs were not high, the evolution of multiple copies of the NCS genes could be important for providing sufficient substrates for subsequent reactions that lead to the biosynthesis of highly cumulative magnoflorine and AAs in A. contorta [11,44,46,48,49].
It has been shown that the Pictet–Spengler reaction can proceed spontaneously in the absence of enzymes [50]. However, the conditions required for such a spontaneous reaction are highly stringent for plants. So far, no evidence has been reported regarding whether this reaction occurs spontaneously in vivo in plants. In order to provide sufficient substrates for subsequent reactions, as mentioned above, BIA-producing plants evolve multiple copies of NCSs. On the other hand, some of the NCS enzymes evolved to possess tandem repeat structures. For instance, P. somniferum NCS has one to four tandem repeats [48]. Among the 15 AcNCSs identified in this study, 8, including AcNCS1–AcNCS8, had one catalytic domain. The other seven, including AcNCS9–AcNCS15, had two catalytic domains. Interestingly, an enzyme catalytic activity assay shows that AcNCS14 and AcNCS15, which have two catalytic domains, exhibit higher catalytic efficiency than AcNCS2, AcNCS4, and AcNCS5, which have a single catalytic domain. This indicates that increasing the number of catalytic domains could enhance the catalytic efficiency of AcNCSs.
NCS enzymes are stereoselective. Ranunculales NCSs specifically catalyze the generation of (S)-norcoclaurine [24,25,27,42,43]. NCSs from N. nucifera of Proteales catalyze the production of almost equal amounts of (S)- and (R)-norcoclaurine [44]. A gymnosperm NCS from G. montanum catalyzes norcoclaurine generation with the majority as (S)-configuration and a minority as (R)-configuration [44]. Precolumn derivatization analysis of the AcNCS-catalyzed products show that there were more (S)-norcoclaurine than (R)-norcoclaurine (Figure 8). Variation in the (S)- and (R)-norcoclaurine proportions could be due to changes in the amino acid residues of the NCS proteins. The key amino acid residues responsible for steroselectivity remain to be identified. In addition, the steroselectivity of NCSs seems to be associated with the substrate specificity of downstream enzymes in the BIA biosynthetic pathway. For instance, CjCYP80G2 from C. japonica exclusively accepts (S)-configured substrates, whereas NnCYP80Q1 from N. nucifera only accepts (R)-configured substrates [51,52]. AcCYP80G7 and AcCYP80Q8 from A. contorta lack stereoselectivity; they can accept (S)- and (R)-configured substrates with equal efficiency [12]. The STORR fusion enzyme in the BIA pathway of Ranunculales can convert (S)-reticuline to (R)-reticuline [37,38]. These results highlight the diversity of stereochemical conversions within the BIA pathway. Currently, the chirality of BIA in A. contorta and the mechanisms underlying stereochemical conversion in A. contorta remain largely unknown, which requires further investigation.

4. Materials and Methods

4.1. Plant Materials and Chemicals

The roots, stems, leaves, and flowers of A. contorta Bunge used for the gene expression profiling and norcoclaurine content determination were collected from the Medicinal Botanical Garden at the Institute of Medicinal Plant Development, Beijing, China. After labeling, the collected plant samples were rapidly frozen in liquid nitrogen and stored at −80 °C until use. The reference standards, including dopamine, 4-hydroxyphenylacetaldehyde (4-HPAA), and (S, R)-norcoclaurine, were purchased from Beijing Xinhuitian Dongfang Biotechnology (Beijing, China), Toronto Research Chemicals (Toronto, ON, Canada), and Chendu Biopurify Phytochemicals (Chengdu, China), respectively.

4.2. AcNCS Gene Identification

The amino acid sequences of the four published NCSs, including TfNCS (ACO90248.1), PsNCS1 (AAX56303.1), PsNCS2 (AAX56304.1), and CjNCS2/CjPR10A (A2A1A1.2), were downloaded from the National Center for Biotechnology Information’s (NCBI) website at https://www.ncbi.nlm.nih.gov accessed on 25 July 2023. Blast analysis of the five proteins against the A. contorta whole genome assembly was carried out using the tBLASTn algorithm with an e-value cut-off of 1 × 10−5 [11,53]. AcNCS gene models were predicted from the retrieved genomic DNA sequences through alignment with NCS genes from other plant species using the BLASTx algorithm with the default parameters (https://blast.ncbi.nlm.nih.gov/Blast.cgi accessed on 28 July 2023). The predicted gene models were further compared with the protein-coding genes annotated for the whole genome assembly of A. contorta through BLASTn analysis [11,53].

4.3. Analyses of Sequence Features, Chromosomal Locations, and Gene Structures

The amino acid compositions, molecular weights, and theoretical isoelectric points were determined using the ProtParam tool (https://web.expasy.org/protparam/ accessed on 13 September 2023). Chromosomal locations of the AcNCS genes were analyzed by comparison analysis of the gene sequences with the whole genome assembly of A. contorta and visualized using TBtools v2.210 [11,54]. Gene structures were determined through comparison of the coding sequence with the whole genome assembly of A. contorta and visualized using TBtools [11,54]. Conservative domains and conservative motifs were analyzed on the Batch-CD (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) and MEME (https://meme-suite.org/meme/ accessed on 5 December 2023) websites, respectively.

4.4. Phylogenetic Tree Construction

Amino acid sequences of 55 NCSs and 11 PR10 proteins from various plants were downloaded from NCBI (Tables S2 and S3). Sequence alignment was performed using ClustalW in MEGA 11 with the default parameters [45]. The neighbor-joining method was utilized to conduct a phylogenetic analysis with a bootstrap value of 1000 replicates [45].

4.5. Quantitative Real-Time Reverse Transcription-PCR (qRT-PCR)

The total RNA was extracted from the roots, stems, leaves, and flowers of A. contorta using the Quick RNA Isolation kit (Huayueyang, Beijing, China). The quality of the extracted RNA was assessed by electrophoresis with 1.2% agarose gel. The concentration of the extracted RNA was determined using a Nanodrop2000 (ThermoFisher Scientific, Waltham, MA, USA). cDNAs were synthesized from 1 μg of total RNA using the TRUEscript 1st Strand cDNA Synthesis kit (Aidlab, Beijing, China) as per the manufacturer’s instructions. Gene expression was determined by qRT-PCR using SYBR Green qPCR Mix (Aidlab, Beijing, China). β-Actin was utilized as the internal reference gene. Three biological and three technical replicates were carried out. Gene-specific primers were designed on the IDT server (https://sg.idtdna.com/scitools/Applications/RealTimePCR/Default.aspx accessed on 10 August 2024). The primers are listed in Table S5. The thermal profile for the qRT-PCR was as follows: pre-denaturation at 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 20 s, and 72 °C for 30 s. Melting curves were obtained by treating the PCR products at 95 °C for 5 s, 65 °C for 5 s, and 95 °C for 15 s. The gene expression levels of the AcNCS genes were calculated using the 2−ΔΔCt method after normalization to the internal reference gene.

4.6. Norcoclaurine Content Determination

The contents of norcoclaurine in the roots, stems, leaves, and flowers were determined using LC-MS/MS. The roots, stems, leaves, and flowers were dried for 24 h in an oven at 37 °C (DH5000II, TAISTE, Tianjing, China). Norcoclaurine was extracted from 0.2 g of roots, stems, leaves, and flowers by ultrasonic extraction with 80% methanol. The LC-MS/MS system, which consisted of an Agilent 1260 Infinity HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with an Agilent Poroshell 120 SB C18 column (100 mm × 2.1 mm, 2.7 µm) and an AB SCIEX 4500 QTRAP MS system (AB SCIEX, Redwood City, CA, USA), was used for the sample analysis. The mobile phase for the gradient elution consisted of (A) water (containing 0.1% formic acid) and (B) acetonitrile with the following gradient procedure: 0–0.3 min, 90% A; 0.3–3 min, 90–2% A; 3–3.5 min, 2% A; 3.5–4 min, 2–90% A; and 4–5 min, 90% A, with a flow rate of 0.25 mL/min. The injection volume was 5.0 µL. Electrospray ionization (ESI) in the positive-ion mode and multiple reaction monitoring (MRM) scanning was employed for the quantification. The curtain gas (CUR), nebulizer gas (GS1), and auxiliary gas (GS2) were set at 15, 40, and 50 psi, respectively. The ion spray voltage (IS) was adjusted to 4000 V for the positive-ion mode, and the source temperature was maintained at 350 °C. Chromatographic workstation (v1.6.2) was used for the data analysis.

4.7. AcNCS Recombinant Protein Expression and Purification

Full-length coding sequences of the AcNCS genes were amplified by PCR using Phanta Flash Master Mix (Vazyme, Nanjing, China). The primers used for cloning are listed in Table S6. The vector pET-30a was linearized by the endonucleases NdeI and NotI. PCR products were subcloned into a pET-30a vector through homologous recombination using the ClonExpress II One-Step Cloning kit (Vazyme, Nanjing, China). The C-terminal of the recombinant protein carries a His label. The cloned sequence was confirmed by sequencing. The vector with a correct sequence was introduced into E. coli strain BL21 (DE3). Cells were incubated at 37 °C in Luria–Bertani (LB) medium until reaching an OD600 of 0.6–0.8. The AcNCS recombinant proteins were induced with 0.5 mmol L-1 IPTG at 16 °C for 20 h. The cells were collected by centrifugation for 10 min at 8000 rpm in 4 °C and then resuspended in 20 mM HEPES buffer I (500 mM of NaCl and 20 mM of imidazole, pH 7.4). After harvesting, the cells were sonicated on ice. Crude AcNCS proteins were purified using a Ni-NTA column (Abbkine, Wuhan, China) with buffer II (20 mM of HEPES, 500 mM of NaCl, and 300 mM of imidazole, pH 7.4). Purified proteins were concentrated and desalted in 20 mM HEPES buffer III (500 mM of NaCl, 20 mM of imidazole, and 10% glycerol, pH 7.4) using an Amicon Ultra-15 mL Centrifugal Filter (Millipore, Burlington, MA, USA). Aliquots were stored at −80 °C. The purity of the recombinant proteins was examined by SDS-PAGE. The protein concentration was quantified using the BCA Protein Assay kit (Takara, Beijing, China).

4.8. In Vitro Enzymatic Activity Assay of AcNCS Recombinant Proteins

The reaction was carried out in a 100 μL reaction system consisting of 50 mM HEPES buffer (pH 7.4). The reaction system contained 2 mM of dopamine, 2 mM of 4-HPAA, 1 mM of ascorbic acid, and 50 μg of proteins. The reaction was conducted at 42 °C for 30 min and then terminated by the addition of 50 μL methanol. The mixture was centrifuged at 13,000 rpm for 10 min, and the supernatant was used for the assay. Reaction products were separated on an ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm) using an ACQUITY UPLC system (Waters, Milford, MA, USA). Samples (2 μL) were eluted with 0.1% formic acid in carbinol (A) and 0.1% formic acid in water (B) based on the following gradient: 0–1 min, 3% A; 1–11 min, 3–97% A; 11–16 min, 97–3% A; and 16–20 min, 3% A. The flow rate was 0.3 mL/min, and the photodiode array (PDA) spectrum was 280 nm. Mass spectrometry detection was performed on an Orbitrap Exploris 120 (ThermoFisher Scientific) mass spectrometer coupled with an Xcalibur in the positive-ion mode based on the information-dependent acquisition mode.

4.9. Kinetic Analysis of AcNCS Recombinant Proteins

The kinetics of the AcNCS recombinant proteins were analyzed in a 100 μL reaction system consisting of 50 mM of HEPES buffer, 50 µg of recombinant proteins, and different concentrations of the substrate. The reaction was carried out at 42 °C for 30 min, and then 100 µL of methanol was added to terminate the reaction. The reaction products were analyzed using a UPLC system, as described in Section 4.8. Enzyme activities were determined by measuring the changes in the substrate content. To determine the kinetic parameters, the saturation concentration of one substrate was set to 2 mM, while the concentration of the other substrate was varied at different levels, including 10, 100, 200, 500, 1000, 2500, and 5000 µM. The kinetic constant of the substrate was determined by monitoring the consumption of the substrate. The enzyme assay was repeated three times at each substrate concentration. The Vmax and Km values were calculated using nonlinear regression analysis with Graph Prism 9.0 software.

4.10. Chiral Analysis of the Configuration of Products

Enzymatic reaction products were extracted with ethyl acetate. Then, the ethyl acetate was evaporated using a nitrogen-blowing instrument. The resulting precipitate was resuspended in 50 μL acetonitrile. An equal volume of 3% pyridine acetonitrile solution and 30 mM R-(-)-DBD-PyNCS acetonitrile were added. After mixing thoroughly, the reaction was allowed to proceed at 60 °C for 1h. The reaction products were detected using an Agilent 1260 liquid chromatography system (Agilent Technologies, Santa Clara, CA, USA) equipped with an Agilent Zorbax Eclipse Plus C 18 column (250 × 4.6 mm, 5 µm). The mobile phase consisted of 0.2% formic acid in water–methanol–acetonitrile (45:30:35, v/v/v) at a flow rate of 0.5 mL/min. The total running time was 35 min, injection volume was 10 μL, flow rate was 0.5 mL/min, and column temperature was 35 °C. Detection was performed using a UV detector at a wavelength of 280 nm.

5. Conclusions

Through genome-wide identification, a total of 15 A. contorta NCSs were identified. The conserved domain analysis showed that eight of them contained one catalytic domain, whereas the other seven had two. The phylogenetic analysis showed that 15 AcNCS proteins can be classified into two clades. The gene expression analysis showed that five AcNCSs exhibited relatively high expression in tissues where norcoclaurine accumulated. The enzyme catalytic activity assay and precolumn derivatization analysis showed that all of the five AcNCSs could catalyze the formation of (S)- and (R)-norcoclaurine, revealing the uniqueness and complexity of the BIA pathway in A. contorta. This is the first report of a functional analysis of AcNCSs. The results provide useful information for manipulating the production of BIAs and their derivatives in A. contorta through genetic and biotechnological approaches and serve as a foundation for the development of low-toxicity or non-toxic medicinal plant resources. Future studies could concentrate on exploring the mechanisms of the stereochemical conversion of BIAs and their derivatives in A. contorta. The downstream pathway leading to AA biosynthesis also requires intensive study.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms26094314/s1.

Author Contributions

Conceptualization, S.L.; investigation, Y.X., S.Z., F.M., W.L. and C.L.; formal analysis, Y.X., S.Z., F.M., W.L., Y.P., B.Z., L.N., C.W. and C.L.; supervision, S.L.; writing—original draft preparation, Y.X.; writing—review and editing, C.L. and S.L.; funding acquisition, S.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the National Natural Science Foundation of China (32370275) and the CAMS Innovation Fund for Medical Sciences (CIFMS) (grant number: 2021-I2M-1-029).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data are available in the article and its Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Kuo, P.C.; Li, Y.C.; Wu, T.S. Chemical constituents and pharmacology of the Aristolochia species. J. Tradit. Complement. Med. 2012, 2, 249–266. [Google Scholar] [CrossRef] [PubMed]
  2. Chawla, P.; Chawla, A.; Shah, G.; Baghel, U.S.; Dhawan, R.K. A review on pharmacognosy and biological activities of Aristolochia. Asian J. Res. Biol. Pharm. Sci. 2013, 1, 101–110. [Google Scholar]
  3. Comission, C.P. Pharmacopoeia of the People Republic of China; China Medical Science Press: Beijing, China, 2015; Volume 1, pp. 51–52. [Google Scholar]
  4. Zhang, C.Y.; Yu, J.; Wang, B. Analysis of volatile oil constituents in the fruits of Aristolochia contorta by GC-MS. Chin. J. Nat. Med. 2004, 2, 126–128. [Google Scholar]
  5. Yuan, J.; Nie, L.; Zeng, D.; Luo, X.; Tang, F.; Ding, L.; Liu, Q.; Guo, M.; Yao, S. Simultaneous determination of nine aristolochic acid analogues in medicinal plants and preparations by high-performance liquid chromatography. Talanta 2007, 73, 644–650. [Google Scholar] [CrossRef]
  6. Tian, H.M.; Cheng, X.M.; Wang, C.H.; Wang, Z.T. Simultaneous determination of four constituents in Aristolochia debilis by HPLC. Chin. Tradit. Pat. Med. 2016, 38, 560–565. [Google Scholar]
  7. Wu, P.L.; Su, G.C.; Wu, T.S. Constituents from the stems of Aristolochia manshuriensis. J. Nat. Prod. 2003, 66, 996–998. [Google Scholar] [CrossRef]
  8. Shi, L.S.; Kuo, P.C.; Tsai, Y.L.; Damu, A.G.; Wu, T.S. The alkaloids and other constituents from the root and stem of Aristolochia elegans. Bioorg. Med. Chem. 2004, 12, 439–446. [Google Scholar] [CrossRef] [PubMed]
  9. Ferreira, M.L.R.; de Pascoli, I.C.; Nascimento, I.R.; Zukerman- Schpector, J.; Lopes, L.M.X. Aporphine and bisaporphine alkaloids from Aristolochia lagesiana var. intermedia. Phytochemistry 2010, 71, 469–478. [Google Scholar] [CrossRef]
  10. Mao, W.W.; Gao, W.; Liang, Z.T.; Li, P.; Zhao, Z.Z.; Li, H.J. Characterization and quantitation of aristolochic acid analogs in different parts of Aristolochiae fructus using UHPLC-Q/TOF-MS and UHPLC-QqQ-MS. Chin. J. Nat. Med. 2017, 15, 392–400. [Google Scholar] [CrossRef]
  11. Cui, X.; Meng, F.; Pan, X.; Qiu, X.; Zhang, S.; Li, C.; Lu, S. Chromosome-level genome assembly of Aristolochia contorta provides insights into the biosynthesis of benzylisoquinoline alkaloids and aristolochic acids. Hortic. Res. 2022, 9, uhac005. [Google Scholar] [CrossRef]
  12. Meng, F.; Zhang, S.; Su, J.; Zhu, B.; Pan, X.; Qiu, X.; Cui, X.; Wang, C.; Niu, L.; Li, C.; et al. Characterization of two CYP80 enzymes provides insights into aporphine alkaloid skeleton formation in Aristolochia contorta. Plant J. 2024, 118, 1439–1454. [Google Scholar] [CrossRef] [PubMed]
  13. Balachandran, P.; Wei, F.; Lin, R.; Khan, I.A.; Pasco, D.S. Structure activity relationships of aristolochic acid analogues: Toxicity in cultured renal epithelial cells. Kidney Int. 2005, 67, 1797–1805. [Google Scholar] [CrossRef]
  14. Grosse, Y.; Baan, R.; Straif, K.; Secretan, B.; El Ghissassi, F.; Bouvard, V.; Benbrahim-Tallaa, L.; Guha, N.; Galichet, L.; Cogliano, V.; et al. A review of human carcinogens-Part A: Pharmaceuticals. Lancet Oncol. 2009, 10, 13–14. [Google Scholar] [CrossRef] [PubMed]
  15. Stiborová, M.; Arlt, V.M.; Schmeiser, H.H. DNA adducts formed by aristolochic acid are unique biomarkers of exposure and explain the initiation phase of upper urothelial cancer. Int. J. Mol. Sci. 2017, 18, 2144. [Google Scholar] [CrossRef] [PubMed]
  16. Chen, L.; Zhang, C.; Xue, R.; Liu, M.; Bai, J.; Bao, J.; Wang, Y.; Jiang, N.; Li, Z.; Wang, W.; et al. Deep whole-genome analysis of 494 hepatocellular carcinomas. Nature 2024, 627, 586–593. [Google Scholar] [CrossRef]
  17. Sahib, S.; Yan, J.; Chen, T. Application of duplex sequencing to evaluate mutagenicity of aristolochic acid and methapyrilene in Fisher 344 rats. Food Chem. Toxicol. 2024, 185, 114512. [Google Scholar] [CrossRef]
  18. Senkin, S.; Moody, S.; Díaz-Gay, M.; Abedi-Ardekani, B.; Cattiaux, T.; Ferreiro-Iglesias, A.; Wang, J.; Fitzgerald, S.; Kazachkova, M.; Vangara, R.; et al. Geographic variation of mutagenic exposures in kidney cancer genomes. Nature 2024, 629, 910–918. [Google Scholar] [CrossRef]
  19. Liang, W.; Xu, Y.; Cui, X.; Li, C.; Lu, S. Genome-wide identification and characterization of miRNAs and natural antisense transcripts show the complexity of gene regulatory networks for secondary metabolism in Aristolochia contorta. Int. J. Mol. Sci. 2024, 25, 6043. [Google Scholar] [CrossRef]
  20. Samanani, N.; Alcantara, J.; Bourgault, R.; Zulak, K.G.; Facchini, P.J. The role of phloem sieve elements and laticifers in the biosynthesis and accumulation of alkaloids in opium poppy. Plant J. 2006, 47, 547–563. [Google Scholar] [CrossRef]
  21. Dastmalchi, M.; Park, M.R.; Morris, J.S.; Facchini, P. Family portraits: The enzymes behind benzylisoquinoline alkaloid diversity. Phytochem. Rev. 2018, 17, 249–277. [Google Scholar] [CrossRef]
  22. Lee, E.J.; Facchini, P. Norcoclaurine synthase is a member of the pathogenesis-related 10/Bet v1 protein family. Plant Cell 2010, 22, 3489–3503. [Google Scholar] [CrossRef] [PubMed]
  23. Berkner, H.; Seutter von Loetzen, C.; Hartl, M.; Randow, S.; Gubesch, M.; Vogel, L.; Husslik, F.; Reuter, A.; Lidholm, J.; Ballmer-Weber, B.; et al. Enlarging the toolbox for allergen epitope definition with an allergen-type model protein. PLoS ONE 2014, 9, e11169. [Google Scholar] [CrossRef] [PubMed]
  24. Li, X.; Li, Q.; Jiao, X.; Tang, H.; Cheng, Y.; Ma, Y.; Cui, G.; Tang, J.; Chen, Y.; Guo, J.; et al. Phylogenetic analysis and functional characterization of norcoclaurine synthase involved in benzylisoquinoline alkaloids biosynthesis in Stephania tetrandra. J. Cell. Physiol. 2024, 239, e31065. [Google Scholar] [CrossRef]
  25. Samanani, N.; Facchini, P.J. Purification and characterization of norcoclaurine synthase. J. Biol. Chem. 2002, 277, 33878–33883. [Google Scholar] [CrossRef]
  26. Samanani, N.; Liscombe, D.K.; Facchini, P.J. Molecular cloning and characterization of norcoclaurine synthase, an enzyme catalyz ing the first committed step in benzylisoquinoline alkaloid bio synthesis. Plant J. 2004, 40, 302–313. [Google Scholar] [CrossRef] [PubMed]
  27. Liscombe, D.K.; MacLeod, B.P.; Loukanina, N.; Nandi, O.I.; Facchini, P.J. Evidence for the monophyletic evolution of benzylisoquinoline alkaloid biosynthesis in angiosperms. Phytochemistry 2005, 66, 2501–2520. [Google Scholar] [CrossRef]
  28. Minami, H.; Dubouzet, E.; Iwasa, K.; Sato, F. Functional analysis of norcoclaurine synthase in Coptis japonica. J. Biol. Chem. 2007, 282, 6274–6282. [Google Scholar] [CrossRef]
  29. Pesnot, T.; Gershater, M.C.; Ward, J.M.; Hailes, H.C. The catalytic potential of Coptis japonica NCS2 revealed-development and utilisation of a fluorescamine-based assay. Adv. Synth. Catal. 2012, 354, 2997–3008. [Google Scholar] [CrossRef]
  30. Marques, J.V.; Dalisay, D.S.; Yang, H.; Lee, C.; Davin, L.B.; Lewis, N.G. A multi-omics strategy resolves the elusive nature of alkaloids in Podophyllum species. Mol. Biosyst. 2014, 10, 2838–2849. [Google Scholar] [CrossRef]
  31. Vimolmangkang, S.; Deng, X.; Owiti, A.; Meelaph, T.; Ogutu, C.; Han, Y. Evolutionary origin of the NCSI gene subfamily encoding norcoclaurine synthase is associated with the biosynthesis of benzylisoquinoline alkaloids in plants. Sci. Rep. 2016, 6, 26323. [Google Scholar] [CrossRef]
  32. Lechner, H.; Soriano, P.; Poschner, R.; Hailes, H.C.; Ward, J.M.; Kroutil, W. Library of norcoclaurine synthases and their immobilization for biocatalytic transformations. Biotechnol. J. 2018, 13, 1700542. [Google Scholar]
  33. Liu, T.; Zhang, W.; Wang, S.; Tian, Y.; Wang, Y.; Gao, R.; Chen, S.; Sun, W.; Ma, W.; Xu, Z. Metabolome and transcriptome association study reveals biosynthesis of specialized benzylisoquinoline alkaloids in Phellodendron amurense. Chin. Herb. Med. 2024, 17, 178–188. [Google Scholar] [CrossRef] [PubMed]
  34. Chen, D.X.; Pan, Y.; Wang, Y.; Cui, Y.Z.; Zhang, Y.J.; Mo, R.Y.; Wu, X.L.; Tan, J.; Zhang, J.; Guo, L.A.; et al. The chromosome-level reference genome of Coptis chinensis provides insights into genomic evolution and berberine biosynthesis. Hortic. Res. 2021, 8, 121. [Google Scholar] [CrossRef] [PubMed]
  35. Islam, M.R.; Mahdi, J.G.; Bowen, I.D. Pharmacological importance of stereochemical resolution of enantiomeric drugs. Drug Saf. 1997, 17, 149–165. [Google Scholar] [CrossRef]
  36. Wang, Y.; Ngo, V.A.; Wang, X. Stereoselective recognition of morphine enantiomers by μ-opioid receptor. Natl. Sci. Rev. 2024, 11, nwae029. [Google Scholar] [CrossRef] [PubMed]
  37. Winzer, T.; Kern, M.; King, A.J.; Larson, T.R.; Teodor, R.I.; Donninger, S.L.; Li, Y.; Dowle, A.A.; Cartwright, J.; Bates, R.; et al. Morphinan biosynthesis in opium poppy requires a P450-oxidoreductase fusion protein. Science 2015, 349, 309–312. [Google Scholar] [CrossRef]
  38. Farrow, S.C.; Hagel, J.M.; Beaudoin, G.A.W.; Burns, D.C.; Facchini, P.J. Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy. Nat. Chem. Biol. 2015, 11, 728–732. [Google Scholar] [CrossRef]
  39. Guo, L.; Winzer, T.; Yang, X.; Li, Y.; Ning, Z.; He, Z.; Teodor, R.; Lu, Y.; Bowser, T.A.; Graham, I.A.; et al. The opium poppy genome and morphinan production. Science 2018, 362, 343–347. [Google Scholar] [CrossRef]
  40. Catania, T.; Li, Y.; Winzer, T.; Harvey, D.; Meade, F.; Caridi, A.; Leech, A.; Larson, T.R.; Ning, Z.; Chang, J.; et al. A functionally conserved STORR gene fusion in Papaver species that diverged 16.8 million years ago. Nat. Commun. 2022, 13, 3150. [Google Scholar] [CrossRef]
  41. Diaz-Bárcena, A.; Fernandez-Pacios, L.; Giraldo, P. Structural characterization and molecular dynamics study of the REPI fusion protein from Papaver somniferum L. Biomolecules 2023, 14, 2. [Google Scholar] [CrossRef]
  42. Luk, L.Y.; Bunn, S.; Liscombe, D.K.; Facchini, P.J.; Tanner, M.E. Mechanistic studies on norcoclaurine synthase of benzylisoquinoline alkaloid biosynthesis: An enzymatic Pictet-Spengler reaction. Biochemistry 2007, 46, 10153–10161. [Google Scholar] [CrossRef]
  43. Leng, L.; Xu, Z.; Hong, B.; Zhao, B.; Tian, Y.; Wang, C.; Yang, L.; Zou, Z.; Li, L.; Liu, K.; et al. Cepharanthine analogs mining and genomes of Stephania accelerate anti-coronavirus drug discovery. Nat. Commun. 2024, 15, 1537. [Google Scholar] [CrossRef] [PubMed]
  44. Zhang, L.; Zhang, S.; Liao, L.; Tang, H.; Wang, W.; Yin, F.; Han, L.; Zhu, K.; Liu, Y.; Xu, D.; et al. Discovery, structure, and mechanism of the (R, S)-norcoclaurine synthase for the chiral synthesis of benzylisoquinoline alkaloids. ACS Catal. 2023, 13, 15164–15174. [Google Scholar] [CrossRef]
  45. Tamura, K.; Stecher, G.; Kumar, S.; Battistuzzi, F.U. MEGA11: Molecular evolutionary genetics analysis version 11. Mol. Biol. Evol. 2021, 38, 3022–3027. [Google Scholar] [CrossRef]
  46. Ilari, A.; Franceschini, S.; Bonamore, A.; Arenghi, F.; Botta, B.; Macone, A.; Pasquo, A.; Bellucci, L.; Boffi, A. Structural basis of enzymatic (S)-norcoclaurine biosynthesis. J. Biol. Chem. 2009, 284, 897–904. [Google Scholar] [CrossRef] [PubMed]
  47. Qin, L.; Hu, Y.; Wang, J.; Wang, X.; Zhao, R.; Shan, H.; Li, K.; Xu, P.; Wu, H.; Yan, X.; et al. Insights into angiosperm evolution, floral development and chemical biosynthesis from the Aristolochia fimbriata genome. Nat. Plants 2021, 7, 1239–1253. [Google Scholar] [CrossRef]
  48. Li, J.; Lee, E.J.; Chang, L.; Facchini, P.J. Genes encoding norcoclaurine synthase occur as tandem fusions in the Papaveraceae. Sci. Rep. 2016, 6, 39256. [Google Scholar] [CrossRef] [PubMed]
  49. Michl, J.; Kite, G.C.; Wanke, S.; Zierau, O.; Vollmer, G.; Neinhuis, C.; Simmonds, M.S.; Heinrich, M. LC-MS- and 1H NMR-based metabolomic analysis and in vitro toxicological assessment of 43 Aristolochia species. J. Nat. Prod. 2016, 79, 30–37. [Google Scholar] [CrossRef]
  50. Pesnot, T.; Gershater, M.C.; Ward, J.M.; Hailes, H.C. Phosphate mediated biomimetic synthesis of tetrahydroisoquinoline alkaloids. Chem. Commun. 2011, 47, 3242–3244. [Google Scholar] [CrossRef]
  51. Ikezawa, N.; Iwasa, K.; Sato, F. Molecular cloning and characterization of CYP80G2, a cytochrome P450 that catalyzes an intramolecular C-C phenol coupling of (S)-reticuline in magnoflorine biosynthesis, from cultured Coptis japonica cells. J. Biol. Chem. 2008, 283, 8810–8821. [Google Scholar] [CrossRef]
  52. Menéndez-Perdomo, I.M.; Facchini, P.J. Elucidation of the (R)-enantiospecific benzylisoquinoline alkaloid biosynthetic pathways in sacred lotus (Nelumbo nucifera). Sci. Rep. 2023, 13, 2955. [Google Scholar] [CrossRef] [PubMed]
  53. Altschul, S.F.; Madden, T.L.; Schäffer, A.A.; Zhang, J.; Zhang, Z.; Miller, W.; Lipman, D.J. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 1997, 25, 3389–3402. [Google Scholar] [CrossRef] [PubMed]
  54. Chen, C.; Wu, Y.; Li, J.; Wang, X.; Zeng, Z.; Xu, J.; Liu, Y.; Feng, J.; Chen, H.; He, Y.; et al. TBtools-II: A “one for all, all for one” bioinformatics platform for biological big-data mining. Mol. Plant 2023, 16, 1733–1742. [Google Scholar] [CrossRef] [PubMed]
Ijms 26 04314 g001
Figure 1. Chromosome localization and gene structure of AcNCSs: (a) chromosome localization of AcNCSs; (b) intron–exon structures of AcNCSs. Green boxes represent exons, and black lines represent introns.
Figure 1. Chromosome localization and gene structure of AcNCSs: (a) chromosome localization of AcNCSs; (b) intron–exon structures of AcNCSs. Green boxes represent exons, and black lines represent introns.
Ijms 26 04314 g001
Ijms 26 04314 g002
Figure 2. Phylogenetic relationships among NCS proteins. The neighbor-joining tree was constructed using MEGA 11 [45]. The tree consists of 55 NCSs from 22 species (Tables S2 and S3). The NCSs from Magnoliales are shown in blue. AcNCSs are marked with a five-pointed star.
Figure 2. Phylogenetic relationships among NCS proteins. The neighbor-joining tree was constructed using MEGA 11 [45]. The tree consists of 55 NCSs from 22 species (Tables S2 and S3). The NCSs from Magnoliales are shown in blue. AcNCSs are marked with a five-pointed star.
Ijms 26 04314 g002
Ijms 26 04314 g003
Figure 3. Conserved domains and motifs of AcNCS proteins: (a) conserved domains of AcNCS proteins; (b) distributions of conserved motifs of AcNCSs. Motifs predicted with MEME (https://meme-suite.org/meme/ accessed on 25 October 2024) are represented by boxes. The numbers in the boxes (1–10) represent motif 1–motif 10, respectively. The box size indicates the length of the motif. The motif consensus is shown in Table S4.
Figure 3. Conserved domains and motifs of AcNCS proteins: (a) conserved domains of AcNCS proteins; (b) distributions of conserved motifs of AcNCSs. Motifs predicted with MEME (https://meme-suite.org/meme/ accessed on 25 October 2024) are represented by boxes. The numbers in the boxes (1–10) represent motif 1–motif 10, respectively. The box size indicates the length of the motif. The motif consensus is shown in Table S4.
Ijms 26 04314 g003
Ijms 26 04314 g004
Figure 4. Multiple sequence alignments of AcNCSs, TfNCS, and NnNCS1. The P-loop conserved in PR10/Bet v 1 family proteins is shown [22]. The blue dots represent the four active sites (Tyr, Glu, Lys, and Asp) identified in TfNCS [46].
Figure 4. Multiple sequence alignments of AcNCSs, TfNCS, and NnNCS1. The P-loop conserved in PR10/Bet v 1 family proteins is shown [22]. The blue dots represent the four active sites (Tyr, Glu, Lys, and Asp) identified in TfNCS [46].
Ijms 26 04314 g004
Ijms 26 04314 g005
Figure 5. Analysis of AcNCS gene expression in roots, stems, leaves, and flowers using the qRT-PCR method. The expression level in leaves was arbitrarily set to 1. The levels in other tissues were given relative to this. Error bars represent the standard deviations of the mean value from three biological replicates. ANOVA (analysis of variance) was calculated using Graph Prism 9.0. p < 0.05 was considered statistically significant, which is represented by different letters above the bars. p ≥ 0.05 was considered statistically non-significant, which is represented by identical letters above the bars.
Figure 5. Analysis of AcNCS gene expression in roots, stems, leaves, and flowers using the qRT-PCR method. The expression level in leaves was arbitrarily set to 1. The levels in other tissues were given relative to this. Error bars represent the standard deviations of the mean value from three biological replicates. ANOVA (analysis of variance) was calculated using Graph Prism 9.0. p < 0.05 was considered statistically significant, which is represented by different letters above the bars. p ≥ 0.05 was considered statistically non-significant, which is represented by identical letters above the bars.
Ijms 26 04314 g005
Ijms 26 04314 g006
Figure 6. Analyses of AcNCS expression patterns and norcoclaurine contents in roots, stems, leaves, and flowers. (a) Expression patterns of AcNCSs in roots, stems, leaves, and flowers. The hierarchical clustering is based on the expression of AcNCSs as determined by RNA-seq [11]. (b) Analysis of the norcoclaurine contents in four tissues, including roots, stems, leaves, and flowers of A. contorta. Error bars represent the standard deviations of the mean value from three biological replicates. ANOVA (analysis of variance) was calculated using Graph Prism 9.0. p < 0.05 was considered statistically significant, which is represented by different letters above the bars. p ≥ 0.05 was considered statistically non-significant, which is represented by identical letters above the bars.
Figure 6. Analyses of AcNCS expression patterns and norcoclaurine contents in roots, stems, leaves, and flowers. (a) Expression patterns of AcNCSs in roots, stems, leaves, and flowers. The hierarchical clustering is based on the expression of AcNCSs as determined by RNA-seq [11]. (b) Analysis of the norcoclaurine contents in four tissues, including roots, stems, leaves, and flowers of A. contorta. Error bars represent the standard deviations of the mean value from three biological replicates. ANOVA (analysis of variance) was calculated using Graph Prism 9.0. p < 0.05 was considered statistically significant, which is represented by different letters above the bars. p ≥ 0.05 was considered statistically non-significant, which is represented by identical letters above the bars.
Ijms 26 04314 g006
Ijms 26 04314 g007
Figure 7. Enzymatic reaction of the AcNCSs. (a) Chemical reaction catalyzed by the AcNCSs. (b) UPLC analysis of the products from dopamine and 4-HPPA under the catalysis of AcNCSs. The reaction without AcNCSs was used as the negative control. The reactions of Δ19TfNCS from T. flavum and NnNCS1 from N. nucifera were used as positive controls. (c) MS/MS spectra of norcoclaurine and the products formed under the catalysis of AcNCS.
Figure 7. Enzymatic reaction of the AcNCSs. (a) Chemical reaction catalyzed by the AcNCSs. (b) UPLC analysis of the products from dopamine and 4-HPPA under the catalysis of AcNCSs. The reaction without AcNCSs was used as the negative control. The reactions of Δ19TfNCS from T. flavum and NnNCS1 from N. nucifera were used as positive controls. (c) MS/MS spectra of norcoclaurine and the products formed under the catalysis of AcNCS.
Ijms 26 04314 g007
Ijms 26 04314 g008
Figure 8. Configuration assays of norcoclaurine using precolumn derivatization. The (R, S)-norcoclaurine standard is shown in red. The products from the catalysis of the AcNCSs are shown in blue. The products from the catalysis of NnNCS1 and TfNCS were used as positive controls (pink). The reaction without AcNCS catalysis was used as the negative control (yellow).
Figure 8. Configuration assays of norcoclaurine using precolumn derivatization. The (R, S)-norcoclaurine standard is shown in red. The products from the catalysis of the AcNCSs are shown in blue. The products from the catalysis of NnNCS1 and TfNCS were used as positive controls (pink). The reaction without AcNCS catalysis was used as the negative control (yellow).
Ijms 26 04314 g008
Table 1. Features of AcNCS genes and their deduced proteins.
Table 1. Features of AcNCS genes and their deduced proteins.
Gene NameSequence IDNumber of
Amino Acids		Molecular Weight (kDa)Theoretical Isoelectric Point (pI)Number of IntronsChromosome Localization
AcNCS1EVM0011227.116017.65.121LG02
AcNCS2EVM0014712.116017.64.991LG02
AcNCS3EVM0011942.116418.15.051LG03
AcNCS4EVM0001864.116417.94.911LG02
AcNCS5EVM0010147.116417.95.011LG02
AcNCS6EVM0012460.117318.65.051LG02
AcNCS7EVM0000189.117318.65.051LG02
AcNCS8EVM0011298.119421.35.241LG01
AcNCS9EVM0016647.130333.85.692LG02
AcNCS10EVM0005050.131334.65.002LG02
AcNCS11EVM0007329.131835.56.151LG02
AcNCS12EVM0000274.131835.56.151LG02
AcNCS13EVM0013491.132335.54.632LG01
AcNCS14EVM0002685.135539.05.402LG02
AcNCS15EVM0014575.135539.05.392LG02
Table 2. Kinetic parameters of the recombinant AcNCS proteins toward dopamine.
Table 2. Kinetic parameters of the recombinant AcNCS proteins toward dopamine.
ProteinVmax (µM min−1)Km (µM)Kcat (S−1)Kcat/Km (M−1 S−1)
AcNCS21.6 ± 0.1518.9 ± 109.920.3 ± 1.03.9× 104
AcNCS41.6 ± 0.2531.5 ± 86.521.4 ± 2.34.0 × 104
AcNCS51.6 ± 0.2534.9 ± 78.321.3 ± 2.14.0 × 104
AcNCS142.3 ± 0.2420.8 ± 45.922.1 ± 1.35.3 × 104
AcNCS152.2 ± 0.2502.9 ± 114.621.8 ± 1.44.3 × 104
Table 3. Kinetic parameters of the recombinant AcNCS proteins toward 4-HPAA.
Table 3. Kinetic parameters of the recombinant AcNCS proteins toward 4-HPAA.
ProteinVmax (µM min−1)Km (µM)Kcat (S−1)Kcat/Km (M−1 S−1)
AcNCS21.3 ± 0.2522.5 ± 72.613.1 ± 1.22.5 × 104
AcNCS41.3 ± 0.2530.3 ± 76.413.2 ± 2.02.5 × 104
AcNCS51.4 ± 0.2536.4 ± 78.613.2 ± 1.92.5 × 104
AcNCS141.5 ± 0.2495.3 ± 98.214.6 ± 1.22.9 × 104
AcNCS151.5 ± 0.2506.7 ± 63.814.2 ± 1.42.8 × 104
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Xu, Y.; Zhang, S.; Meng, F.; Liang, W.; Peng, Y.; Zhu, B.; Niu, L.; Wang, C.; Li, C.; Lu, S. Genome-Wide Identification and Functional Analysis of the Norcoclaurine Synthase Gene Family in Aristolochia contorta. Int. J. Mol. Sci. 2025, 26, 4314. https://doi.org/10.3390/ijms26094314

AMA Style

Xu Y, Zhang S, Meng F, Liang W, Peng Y, Zhu B, Niu L, Wang C, Li C, Lu S. Genome-Wide Identification and Functional Analysis of the Norcoclaurine Synthase Gene Family in Aristolochia contorta. International Journal of Molecular Sciences. 2025; 26(9):4314. https://doi.org/10.3390/ijms26094314

Chicago/Turabian Style

Xu, Yayun, Sixuan Zhang, Fanqi Meng, Wenjing Liang, Yunliang Peng, Butuo Zhu, Lili Niu, Chunling Wang, Caili Li, and Shanfa Lu. 2025. "Genome-Wide Identification and Functional Analysis of the Norcoclaurine Synthase Gene Family in Aristolochia contorta" International Journal of Molecular Sciences 26, no. 9: 4314. https://doi.org/10.3390/ijms26094314

APA Style

Xu, Y., Zhang, S., Meng, F., Liang, W., Peng, Y., Zhu, B., Niu, L., Wang, C., Li, C., & Lu, S. (2025). Genome-Wide Identification and Functional Analysis of the Norcoclaurine Synthase Gene Family in Aristolochia contorta. International Journal of Molecular Sciences, 26(9), 4314. https://doi.org/10.3390/ijms26094314

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop