Functional Characterization of a Novel Tropinone Reductase-Like Gene In

Home , Tropinone reductase I, Tropinone reductase II

Journal of Plant Physiology 170 (2013) 958–964

Contents lists available at SciVerse ScienceDirect

Journal of Plant Physiology

journa l homepage: www.elsevier.com/locate/jplph

Functional Biotechnology

Functional characterization of a novel tropinone reductase-like gene in

Dendrobium nobile Lindl

a,1 a,1 a b a,∗

Xiaofei Cheng , Wei Chen , Zhenhua Zhou , Junjun Liu , Huizhong Wang

College of Life and Environmental Science, Hangzhou Normal University, 310036 Hangzhou, China

Paciﬁc Forestry Centre, Canadian Forest Service, Natural Resources Canada, Victoria, V8Z 1M5, Canada

a r a

t i c l e i n f o b s t r a c t

Article history: Dendrobium nobile, a herbal medicine plant, contains many important alkaloids and other secondary

Received 21 September 2012

metabolites with pharmacological and clinical effects. However, the biosynthetic pathway of these sec-

Received in revised form 31 January 2013

ondary metabolites is largely unknown. In present study, a cDNA sequence (DnTR2) that encodes a

Accepted 5 February 2013

peptide with high similarity to known tropinone reductase (TR) was cloned from D. nobile Lindl. Sequence

Available online 6 April 2013

comparison and phylogenetic analysis showed that DnTR2 was evolutionarily distant from those well-

characterized subgroups of TRs. qRT-PCR revealed that DnTR2 was expressed constitutively in all three

Keywords:

vegetative organs (leaves, stems and roots) and was regulated by methyl jasmonate (MeJA), salicylic acid

Dendrobium nobile

(SA) and nitrogen oxide (NO). Catalytic activity analysis using recombinant protein found that DnTR2

Functional analysis

was not able to reduce tropinone, but reduced the two structural analogs of tropinone, 3-quinuclidinone

Secondary metabolism

Substrate speciﬁcity hydrochloride and 4-methylcyclohexanone. Structural modeling and comparison suggested that the sub-

Tropinone reductase strate speciﬁcity of TRs may not be determined by their phylogenetic relationships but by the amino acids

that compose the substrate binding pocket. To verify this hypothesis, a site-directed mutagenesis was

performed and it successfully restored the DnTR2 with tropinone reduction activity. Our results also

showed that the substrate speciﬁcity of TRs was determined by a few residues that compose the sub-

strate binding pocket which may have an important role for directed selecting of TRs with designated

substrate speciﬁcities.

Introduction Tropinone reductases (TRs), members of the SDR superfamily,

use the NADPH as coenzyme to reduce tropinone. Previous stud-

Short chain dehydrogenase/reductase (SDR) superfamily is a ies showed that there were two kinds of TRs, namely TRI and

group of NAD(P)H-dependent oxidoreductases that typically con- TRII, which differed from each other by the stereo speciﬁcities

sist of 250–350 amino acid residues (Oppermann et al., 2003). SDRs of their reduction products. TRI reduces tropinone into tropine,

differ greatly in the substrate speciﬁcity and subsequently in their the pre-product of hyoscyamine, whereas TRII reduces tropinone

function. However, SDRs have very similar three dimensional struc- into pseudotropine (Nakajima et al., 1998). TRs were found mainly

tures with two conserved domains, an N-terminal core domain and in plants within or closely related to the family of Solanaceae

a C-terminal domain. The N-terminal core domain, which includes (Hashimoto et al., 1992; Kai et al., 2009; Kaiser et al., 2006;

most of the polypeptide, adopts a Rossmann fold and contains a Keiner et al., 2002; Nakajima et al., 1993; Portsteffen et al., 1994;

conserved G-X3-G-X-G motif for NAD(P)H-binding (Persson et al., Rothe and Dräger, 2002). Recently, TR homologues were also

1991). The C-terminal domain, which protrudes from the core found in other plant families, such as Arabidopsis thaliana, Brachy-

domain, contains several amino acids that are crucial for substrate podium distachyon, Vitis vinifera, and Cochlearia ofﬁcinalis. Of these

binding (Oppermann et al., 2003). SDR binds its substrate at the TR homologues, only CoTR from C. ofﬁcinalis (a species of the

deep cleft between the N-terminal core domain and the small lobe. Brassicaceae) has been proven with tropinone reduction activity

(Brock et al., 2008), whereas Arabidopsis AtSDR does not process a

tropinone reduction activity (Brock et al., 2008). However, some TR

homologues (such as AtSDR) are functional reductases with other

Abbreviations: DnTR2, D. nobile tropinone reductase 2; IPTG, isopropyl-

␤-D-1-thiogalactopyranoside; MeJA, methyl jasmonate; qRT-PCR, quantitative substrates than tropinone (Brock et al., 2008), implying functional

reverse-transcriptase-polrmerase chain reaction; SA, salicylic acid; SDR, short-chain

divergence of TR-like short-chain dehydrogenases in higher plants.

dehydrogenase; SNP, sodium nitroprusside; TR, tropinone reductase.

∗ Dendrobium nobile, a species of important herbal medicine plant,

Corresponding author. Tel.: +86 571 2886 5330; fax: +86 571 2886 5330.

contains many medically important secondary metabolic compo-

E-mail address: [email protected] (H. Wang).

1 nents, e.g. dendrobine and bioactive polysaccharides (Luo et al.,

These authors contributed equally to this work.

X. Cheng et al. / Journal of Plant Physiology 170 (2013) 958–964 959

2009; Wang et al., 2010), and has been developed into many prod- concentrated with an Amicon Ultra-15 Centrifugal Filter Unit (Mil-

ucts for health care. However, little information is available about lipore, Billerica, MA, USA) to a desired concentration; and stored at

− ◦

the genes that are involved in the secondary metabolism of D. 80 C.

nobile at present. Previously, we constructed a cDNA library of D.

nobile to assist cloning the genes that may function in D. nobile

Protein blot analysis

secondary metabolism (Chen et al., 2013). During analysis the

expressed sequence taqs (ESTs) of D. nobile, we identiﬁed several

Protein blot analysis was performed as described previously

cDNA fragments that encode peptides that share high sequence

using antibodies against histidine tag (Zuoyang Bio., Beijing, China)

similarities with putative TRs encoded by B. distachyon, V. vinifera

(Chen et al., 2013).

and A. thaliana. One of the TR-like genes of D. nobile (DnTR1) encodes

a functional TR enzyme that is able to reduce tropinone into tropine

Site-directed mutagenesis

(Chen et al., 2013). In order to understand diversiﬁcation of the SDR

superfamily, in the present study we further cloned and function- ®

The mutagenesis was performed using a QuikChange Site-

ally characterized another D. nobile TR-like gene (named as DnTR2).

Directed Mutagenesis Kit (Stratagene). In detail, the pET:DnTR2

plasmid, that contain wild type of DnTR2, was ampliﬁed using the

Materials and methods

oligonucleotides DnTR2-Y201V-D (5 -ATCAATTCTG TTTCACCATG

GGTAATCAAG ACTTCACTCG TAAAC-3 ) and DnTR2-Y201V-R (5 -

Plant growth conditions

GTTTACGAGT GAAGTCTTGA TTACCCATGG TGAAACAGAA TTGAT-

3 ) which were designed to mutate the Tyr-201 (TAT) to Valine

Dendrobium nobile plants were grown in moss media under the

◦ ◦ (GTA). Plasmid that contained the designed mutation was veri-

temperature of 22 C during the day and 18 C during the night with

ﬁed by DNA sequencing and transformed into E. coli BL21 (DE3) for

the relative humidity and photoperiod set to 75% and 12:12 (day:

protein over-expression. Protein expression and puriﬁcation were

night), respectively.

performed as described above for wild-type DnTR2.

External plant hormones treatments

Enzyme assays

For plant hormones treatment experiments, six-months-old

Enzyme activity was assayed as described previously with a few

plants were selected and sprayed with 100 mM methyl jasmonate

modiﬁcations (Hashimoto et al., 1992). In detail, enzymatic assay

(MeJA), 1% (m/v) salicylic acid (SA), 100 mM sodium nitroprusside ◦

was performed at 25 C in a total of 1 mL 100 mM sodium phos-

(SNP; exogenous NO donor), or distilled water as a negative control

phate (pH 8.0) reaction buffer that contained 1–400 ␮g protein,

for 1 min. After the treatment, plants were transferred to a growth

200 mM NADPH, 5 mM substrate (0.01–25 mM for determination of

chamber with the same growth conditions. Leaves, stems, and roots

KM values). Three substrates, including tropinone (Sigma–aldrich,

were collected separately at 0, 4, 8, 16, and 24 h post each treatment,

St. Louis, MO, USA), 3-quinuclidinone hydrochloride (Damas-beta,

and frozen in liquid nitrogen for further analysis. Each treatment

Basel, Switzerland), and 4-methylcyclohexanone (Sigma–aldrich)

contained 12 plants with three biological repeats.

were tested. As negative control, boiled proteins were added

instead of native DnTR2. All these assays were repeated three times.

Cloning of DnTR2

The kinetic constant of the enzyme was determined using a non-

linear regression of the Michaelis–Menten equation (Kou et al.,

Total RNA was isolated from D. nobile plant using an

2005) using Sigma Plot enzyme kinetics module 9.0.

AxyPrep Multisource Total RNA Miniprep Kit (Axygen Biosciences,

Hangzhou, China) following the manufacturer’s instructions. The

TM qRT-PCR analysis

ﬁrst strand of cDNA was synthesized using an iScript cDNA syn-

thesis Kit (Bio-Rad, Hercules, CA, USA) with Oligo dT(18) as primer.

qRT-PCR was carried out to investigate DnTR2 transcript expres-

cDNA was used as template to amplify the full-length coding region

sion in roots, stems and leaves of D. nobile plants as well as the

of DnTR2 with the primer set: TR1F (5 - ATG GCC GGA GGA GGA GG-

transcriptional response to the plant hormones as described pre-

3 ) and TR1R (5 - TCA AGC ACC TAA GGT CCT-3 ), designed according

viously (Chen et al., 2013) on a CFX96 Real-Time PCR detection

to EST sequence data from a CDNA library. The ampliﬁed frag-

System. PCR mixtures were prepared using an iQ SYBR Green

ment was cloned into the pMD18-T vector (TaKaRa, Dalian, China)

Supermix kit (Bio-Rad) with the primer set QTR2F (5 -CTC TCG

and recombinant clone was named as pMD:DnTR2. Its nucleotide

GGC GTA TCG GTG AG-3 ) and QTR2R (5 -TCT TCA ACA ATG GCA

sequence was deposited in GenBank under the accession number

◦

TAC CC-3 ). The ampliﬁcation condition was set as follow: 95 C of JQ063459.

◦ ◦

denaturation for 20 s, 40 cycles of 95 C denaturation for 15 s, 58 C

◦

annealing for 15 s, and 72 C elongation for 15 s. As an internal con-

Recombinant protein expression and puriﬁcation

trol for normalization of DnTR2 expression levels, a 196 bp fragment

of D. nobile actin gene (DnActin7, GenBank acc no. JQ040497) was

The DnTR2 coding region was ampliﬁed from pMD:DnTR2

ampliﬁed as using primers set actinF (5 -GAT GTG CAG AGG TGC

with the primer set of pTR1F (5 -CAT ATG GCC GGA GGA GGA

TTT TC-3 ) and actinR (5 -GCT TCT CCT TGA TAT CTC GAA-3 ). Each

GG-3 ) (NdeI site underlined) and pTR1R (5 -CTC GAG TCA AGC

sample was run in triplicate.

ACC TAA GGT CCT-3 ) (XhoI site underlined) and inserted into

the pET-28a vector (Novagen, Darmstadt, Germany). The result-

ing plasmid, pET:DnTR2, was used for protein expression. DnTR2 Sequence analysis

was expressed as a fusion peptide with N-terminal 6 × His-tag

◦

in E. coli strain BL21(DE3) at 37 C for 4 h with the presence DNAMAN version 6.0.3.40 software (Lynnon Corporation, QC,

␤

of 1 mM isopropyl- -D-1-thiogalactopyranoside (IPTG). Recombi- Canada) was used for protein sequences assembling. Phylogenetic

nant DnTR2 was puriﬁed as described previously (Cheng et al., tree was constructed with the neighbor joining method using

2011). Protein concentration was determined using a Bio-Rad pro- MEGA 5.0 software (Tamura et al., 2007). The Bootstrap conﬁdence

tein assay kit I (Bio-Rad) as instructed. Puriﬁed protein was ﬁnally limits were obtained by 1000 replicates.

960 X. Cheng et al. / Journal of Plant Physiology 170 (2013) 958–964

Structural modeling and analysis theoretical pI of 6.44. DnTR2 amino acid sequence contained a

conserved Rossmann folding structure, which includes a con-

A three-dimensional model of DnTR2 was constructed by the served NAD(P)H binding motif (Gly-X3-Gly-X-Gly) and catalytic

SWISS-MODEL server using the crystal structure of TRII encoded residues Ser-Asn-Lys, suggesting that DnTR2 is a member of the

by Datura stramonium (DsTRII, PDB ID: 2AE1) as a template (Arnold SDR superfamily. Blastp search showed that DnTR2 has highly

et al., 2006). The NADPH and tropinone were transferred from the sequence identities with several predicted tropinone reductase

template structure by super-positioning the backbone structures homologues, including the homologues encoded by B. distachyon

and subsequent energy minimization. The protein–ligand interac- (XP 003558258), V. vinifera (XP 002277835) and A. thaliana

tion energy was estimated by X-SCORE (Wang et al., 2002). The (NP 196225).

analysis and representation of the electrostatic surface potentials of An unrooted phylogenetic tree was constructed to insight the

TRs were performed by using PyMOL 0.99 software (DeLano, 2002). phylogeny relationship of DnTR2 with other TRs. As shown in Fig. 1,

all TRs that isolated from plants belonging or closely related to

Results the family of Solanaceae were clustered into two groups (TRI and

TRII). TRI group contains the TRs that have activity to convert tropo-

nine into tropine, while TRII included those TRs which can reduce

Cloning and phylogenetic analysis of DnTR2

tropinone into pseudotropine. However, the TRs that isolated from

plants outside the family Solanaceae showed remarkable phylo-

Based on in silico EST assembly of a D. nobile cDNA library,

genetic distances from both TRIs and TRIIs. Among them, DnTR1,

the primer set TR2F and TR2R was designed and used to amplify

CsTRI, and CoTR can accept tropinone as their substrate, whereas

the complete coding region of DnTR2. Sequence analysis showed

AtSDR does not process a reduction activity with tropinone as sub-

that the DnTR2 amino acid coding region was 813 bp in length

strate (Brock et al., 2008; Chen et al., 2013). Interestingly, CoTR

and encoded a peptides consisting of 271 amino acids. The puta-

can catalyze troponine into both tropine and pseudotropine (Brock

tive DnTR2 protein has a molecular mass of 29419.71 Da and a

Fig. 1. Unrooted phylogenetic tree of tropinone reductases. Phylogenetic tree was constructed based on amino acid sequence alignment analysis of putative plant

tropinone reductases. Branch numbers represent a percentage of the bootstrap values in 1000 sampling replicates and the scale bar indicates branch lengths. Sequence

abbreviations: AaTRI and AaTRII, Anisodus acutangulus tropinone reductase I and II (EU424321, EU424322); HnTRI and HnTRII, Hyoscyamus niger tropinone reduc-

tase I and II (D88156, L20485); DsTRI and DsTRII, Datura stramonium tropinone reductase I and II (L20473, L20474); StTRI and StTRII, Solanum tuberosum tropinone

reductase I and II (AJ307584, AJ292343); SdTRII, Solanum dulcamara tropinone reductase II (AM947940); CoTR, Cochlearia ofﬁcinalis tropinone reductase (AM748271);

Os03g0268900, Os03g0269000 and Os11g0438700, Oryza sativa putative tropinone reductases; Sb01g039880 and Sb05g004530 Sorghum bicolor putative tropinone reduc-

tases; Loc100282692 Zea mays putative tropinone reductase; LOC100837602, LOC100838215 and LOC100837309 Brachypodium distachyon putative tropinone reductases;

Phypadraft 227898 Physcomitrella patens putative tropinone reductase; Selmodraft 444601 and Selmodraft 416905, Selaginella moellendorfﬁi putative tropinone reductases;

At5g06060, At2g29290, At2g29360(AtSDR), At2g29150, At1g07440, At2g29370, At1g07450, At2g29330, At2g29340, At2g29290, At2g29310, At2g29320, At2g29300 and

At2t29350 Arabidopsis thaliana putative tropinone reductases.

X. Cheng et al. / Journal of Plant Physiology 170 (2013) 958–964 961

Enzymatic activity analyses of recombinant DnTR2

For biochemical and enzymatic analysis, recombinant DnTR2

protein was expressed in E. coli and puriﬁed to homogeneity

by Ni-NTA afﬁnity chromatography and gel ﬁltration as judged

by SDS-PAGE (Fig. 3A). Identity and integrity of puriﬁed pro-

tein was further conﬁrmed by protein blot analysis using the

antibodies against 6 × histidine tag (Fig. 3B). Puriﬁed DnTR2

was then incubated with NADPH and each of three different

substrates tropinone and tropinone analogs (3-quinuclidinone

hydrochloride and 4-methylcyclohexanone). The reduction veloc-

ity (Vmax) of DnTR2 toward 3-quinuclidinone hydrochloride and

4-methylcyclohexanone were detected as 1.76 ± 0.12 nkat/mg

(Km = 3.1 ± 0.295 mM) and 14.67 ± 2.01 nkat/mg (Km = 7.72 ±

1.91 mM), respectively (Table 1). However, DnTR2 did not show

any reducing activity when tropinone was used as substrate.

Structural comparison of DnTR2 and its homologues

In order to get further insight into the structural differences

that impact on the enzymatic activities, we modeled the three-

dimensional structure of DnTR2 and compared it with the crystal

structures of DsTRI and DsTRII that are encoded by Datura stra-

monium and the modeled structure of DnTR1. Crystal structure of

DsTRI and DsTRII revealed that most of the amino acids for the sub-

strate binding pockets are hydrophobic and localized at the deep

cleft between the core domain and the small lobe (Nakajima et al.,

1998). However, His-112 in DsTRI and Glu-156 in DsTRII are basic

and acidic, respectively (Nakajima et al., 1998). These structural

features causes different orientation of tropinone in the binding

Fig. 2. Transcript expression proﬁles of DnTR2. (a) Expression level of DnTR2 in the

pocket and consequently results in different products in the reduc-

roots, stems and leaves of D. nobile under normal condition. (b) The transcript level

of DnTR2 after MeJA, SNP and SA treatment at 0 h, 4 h, 8 h, 16 and 24 h. Bars represent ing reaction (Nakajima et al., 1998).

the standard deviations of three biological repeats. Although DnTR2 shared only 57.3%, 44.7%, and 43.7% amino acid

sequence identities with DnTR1, DsTRI, and DsTRII, respectively,

the overall ␣-carbon chains of these four proteins are almost iden-

et al., 2008). In this phylogenetic tree DnTR2 formed a separate evo-

tical (Fig. 4A). The inner molecular surfaces of the DnTR2 substrate

lutional branch with a TR-like gene from A. thaliana (At5g06060),

binding pocket was also positively charged due to presence of Arg-

far away from DnTR1 (Fig. 1), suggesting that DnTR1 and DnTR2

110, which is similar to His-112 in DsTRI (Fig. 4B). Most of other

may have different substrate speciﬁcity.

amino acids that composite the DnTR2 substrate binding pocket

are also hydrophobic, but two hydrophobic amino acids in DsTRI or

Expressional pattern and regulation of DnTR2 transcript

DsTRII were replaced by a polar or uncharged amino acid in DnTR2

(Asn-108 and Tyr-201). In DnTR2, Asn-108 is close to Arg-110 that

qRT-PCR was used to analyze DnTR2 transcript proﬁle in planta.

potentially determines substrate binding orientation in the bind-

As shown in Fig. 2A, DnTR2 transcript was detected in all three

ing pocket. However, DnTR2 Tyr-201 is located at the opposite side

tested vegetative organs, including roots, stems, and leaves, indi-

of Arg-110 in the inner substrate binding surface (Fig. 4B). These

cating that DnTR2 is a functional gene with ubiquitous expression

structural characters suggest that the disabled tropinone reduc-

in all tissues. We also tested whether DnTR2 expression is regulated

tion activity of DnTR2 may be caused by the replacement of an

by plant hormones. Three types of hormones, NO, SA and JA, were

uncharged amino acid at position 201.

selected to measure their effects on DnTR2 transcript expression.

These hormones were selected since they have been well charac-

terized as inducers to promote the biosynthesis of plant secondary

Site-directed mutagenesis of DnTR2

metabolites (Memelink et al., 2001; Sharma et al., 2011; Zhang

and Memelink, 2009). As shown in Fig. 2B, the expression level of

In order to conﬁrm the role of Tyr-201 in substrate bind-

DnTR2 transcript increased greatly after each of three treatments.

ing, the DnTR2 Tyr-201 was mutated to Valine in the mutated

Responses of DnTR2 expression to these hormones were different

enzyme (DnTR2-Y201V). Following over-expression in E. coli

in timing and magnitude. MeJA showed the strongest effect on

and puriﬁcation, the puriﬁed DnTR2-Y201V was tested with

DnTR2 transcript expression and caused about a 22-fold (P < 0.001)

NADPH and tropinone. The enzyme activity was then quan-

increase, whereas SA and NO resulted in 16- (P < 0.01) and 9-fold

tiﬁed by measuring the consumption of NADPH at 340 nm.

(P < 0.01) enhancement of DnTR2 transcript, respectively. However,

Surprisingly, tropinone was reduced into tropine by DnTR2-

DnTR2 responded more quickly to SA (8-h) than to NO and JA (16-

Y201V with a reduction velocity (Vmax) of 0.54 ± 0.01 nkat/mg

and 24-h, respectively). These results indicate that DnTR2 may be

(Km = 57.76 ± 0.86 mM). This result indicates that alternation of an

an important reductase functioning in D. nobile defense against

amino acid in the substrate binding pocket can change the enzyme

both biotic and abiotic stresses, since these plant hormones have

substrate speciﬁcity. Furthermore, DnTR2-Y201V remains to have

signaling roles in plant responses to various environmental factors

the ability to reduce the 3-quinuclidinone hydrochloride and 4-

(Hwang et al., 2011; Park et al., 2007; Schaller and Stintzi, 2008;

methylcyclohexanone, although the Vmax of DnTR2-Y201V toward

Zhang and Memelink, 2009).

4-methylcyclohexanone and 3-quinuclidinone hydrochloride and

962 X. Cheng et al. / Journal of Plant Physiology 170 (2013) 958–964

Fig. 3. Expression and puriﬁcation of recombinant DnTR2. (a) Expression of DnTR2 in E. coli. M, Protein molecular weight standard marker (Bio-Rad). Lane 1, E. coli cells

harboring pET:DnTR2 plasmid before IPTG induction; Lane 2, E. coli cells harboring pET:DnTR2 plasmid after IPTG induction. Lane 3, puriﬁed DnTR2 protein. (b) Protein blot

analysis of puriﬁed DnTR2 using monoclonal antibodies against the 6 × his tag. Samples were loaded as the same order in Fig. 3A.

Table 1

Kinetic properties of DnTR2 and DnTR2-Y201V.

−1 −1

Protein Substrate Vmax (nkat/mg) Km (mM) Kcat/Km [sec × M ]

DnTR2

Tropinone 0 – –

4-methylcyclohexanone 14.67 ± 2.01 7.72 ± 1.91 64.55

3-quinuclidinone hydrochloride 1.76 ± 0.12 3.01 ± 0.29 319.37

DnTR2-Y201V

Tropinone 0.54 ± 0.0099 57.75 ± 0.86 0.319

4-methylcyclohexanone 0.63 ± 0.013 2.71 ± 0.42 7.9

3-quinuclidinone hydrochloride 0.52 ± 0.037 2.01 ± 0.55 6.08

Note: Data are the means of three replicates ± standard deviation.

Fig. 4. Structural comparison of TRs encoded by Dendrobium nobile and Datura stramonium. (a) Overall structural comparison of the DnTR2 and DnTR1, DsTRI and DsTRII.

The cartoon loop representation was prepared by the software Pymol. DnTR2, DnTR1, DsTRI, and DsTRII are shown as cyan, yellow, green and pink, respectively. Tropinone

is shown in cyan, and NADPH is shown in red. The N- and C-terminal are also indicated. (b) Local comparison of the active center of DnTR2, DnTRI, DsTRI, and DsTRII. DnTR2

and DnTR1 and DsTRI and DsTRII are shown as cyan, yellow, green and pink, respectively. Tropinone is shown in cyan, and NADPH is shown in red. Amino acid side chains

that form the tropinone binding pocket of DnTR2 are labeled. The amino acids that contribute to the positive and negative electrostatic potential in DsTRI (His 112) and

DsTRII (Glu 156) are labeled. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

X. Cheng et al. / Journal of Plant Physiology 170 (2013) 958–964 963

Fig. 5. Amino acid sequence alignments of the TR regions surrounding the putative tropinone binding pockets. Amino acids predicted to be in contact with the tropinone

substrate are shown in larger uppercase type. Basic and acid amino acids are shown in blue and red background, respectively. Hydrophobic and polar amino acids are shown

in cyan and pink background, respectively. The abbreviations for TR proteins are the same as in Fig. 1. (For interpretation of the references to color in this ﬁgure legend, the

reader is referred to the web version of this article.)

were signiﬁcantly lower (23- and 3-fold decrease, respectively) Tropinone reductase homologous genes are present in many

than that of wild-type DnTR2 protein (Table 1). plant species, nevertheless TRs with tropinone reduction activ-

ity have mainly been found in plants belonging or closely related

to the family of Solanaceae (Dräger, 2006; Grifﬁn and Lin, 2000;

Nakajima et al., 1999). Thus far, only two TR homologous that

Discussion encoded by plants outside the family of Solanaceae, CoTR encoded

by C. ofﬁcinalis and DnTR1 encoded by D. nobile, have been found

Dendrobium, a huge genus of orchids, contains many important to have tropinone reductase activities (Brock et al., 2008; Chen

herbal medicine species, such as D. nobile, D. ofﬁcinale, D. ﬁmbi- et al., 2013). Furthermore, CoTR can reduce tropinone into both

ratum, D. chrysanthum and D. loddigesii. Several economically or tropine and pseudotropine (Brock et al., 2008). Interestingly, as

medically important secondary metabolites, such as dendrobine a phylogenetic neighbor of CoTR, AtSDR (AY090333) does not

(Wang et al., 2010), bioactive polysaccharides (Luo et al., 2009), and have any tropinone reduction activity (Brock et al., 2008). The

bibenzyl phenanthrene (Yang et al., 2006), have been discovered enzymatic assay in present study showed that DnTR2 does not

and characterized in Dendrobium. However, biosynthesis pathways have the tropinone reductase activity. However, replacing the

of these secondary metabolites are still unknown at present. Anal- residue Tyr-201 by Valine rescued the tropinone reduction activ-

ysis of D. nobile ESTs showed that the D. nobile genome encodes ity of DnTR2, suggesting that the tropinone reduction activity

several TR-like genes (data not shown), suggesting that these TR- of a TR-like protein cannot be predicted by its phylogenetic

like genes may have important roles in secondary metabolism in D. relationship with other well-characterized TRs, and it is deter-

nobile. In a previous work we characterized a D. nobile TR-like gene mined by a few amino acids that composite the substrate binding

(DnTR1) and recombinant DnTR1 protein has the ability to reduce pocket.

the tropinone into tropine (Chen et al., 2013). In present study, we To further address this hypothesis, we compared the 11 amino

characterized another D. nobile TR-like gene DnTR2 that is distinct acids (named as site S1 to S11, respectively) that composite the

from DnTR1 in several aspects. The peptides encoded by these two substrate binding pocket of TRs and their homologues. As shown

TRs only share 57.3% identity and are located in separate phylo- in Fig. 5, most amino acids that composite the substrate bind-

genetic branches (Fig. 1). DnTR1 and DnTR2 also differed in their ing pocket are hydrophobic. However, several sites were observed

transcript expression responsive to different hormones. The mRNA being replaced by amino acids with other characteristics. In TRIs, a

expression of DnTR1 is only enhanced at the presence of MeJA (Chen basic residue (His) was localized at S2. This residue was thought to

et al., 2013), whereas the DnTR2 transcript expression is responsive be important for ﬁxing the orientation of tropinone in the substrate

to all three tested plant hormones JA, SA, or NO (Fig. 2), implying binding pocket, and subsequently determines the stereoisomeric

that DnTR1 and DnTR2 may have different physiological roles in speciﬁcity of its reducing product (Nakajima et al., 1998). In TRIIs,

plant defense to environmental stresses. Although both DnTR1 three sites, S2, S4 and S6, are occupied by non hydrophobic residues.

and DnTR2 enzymes can reduce 3-quinuclidinone hydrochloride In these three sites, the acid residue (Glu) at S6 was thought

and 4-methylcyclohexanone, DnTR1 can also accept tropinone as to play key role in ﬁxing tropinone orientation. The other two

its substrate (Chen et al., 2013) whereas DnTR2 cannot reduce residues (Tyr and Ser) at S2 and S4 are polar and uncharged amino

tropinone (Table 1), suggesting the tremendous variations in the acids (Nakajima et al., 1998). The substitution of the hydropho-

substrate speciﬁcity among D. nobile TR-like proteins. bic amino acids at site S2 and S4 by polar and uncharged amino

964 X. Cheng et al. / Journal of Plant Physiology 170 (2013) 958–964

acids seem to be tolerable for maintaining tropinone reduction DeLano WL. The PyMOL molecular graphics system. San Carlos, CA, USA: DeLano

Scientiﬁc; 2002.

activity, since obvious reduction activities can be observed in TRIIs

Dräger B. Tropinone reductases, enzymes at the branch point of tropane alkaloid

when tropinone were used as substrate. However, these replace-

metabolism. Phytochemistry 2006;67:327–37.

ment may have a dose effect on their reduction activity, since TRIs Grifﬁn WJ, Lin GD. Chemotaxonomy and geographical distribution of tropane alka-

loids. Phytochemistry 2000;53:623–37.

always have higher tropinone reducing velocity than TRIIs (Dräger,

Hashimoto T, Nakajima K, Ongena G, Yamada Y. Two tropinone reductases with dis-

2006). There is a wide range of amino acid variations in the sub-

tinct stereospeciﬁcities from cultured roots of hyoscyamus niger. Plant Physiol

strate binding pockets of the TR homologues. Comparison of the TR 1992;100:836–45.

Hwang IS, An SH, Hwang BK. Pepper asparagine synthetase 1 (CaAS1) is required

homologues with tropinone reduction activities (such as CoTR and

for plant nitrogen assimilation and defense responses to microbial pathogens.

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Plant J 2011;67:749–62.

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or both of the site S2 and S4 that functions as key residue to ﬁx tion of two tropinone reductases in anisodus acutangulus and enhancement of

tropane alkaloid production in AaTRI-transformed hairy roots. Biotechnol Appl

tropinone in TRIs or TRIIs are always occupied by non-hydrophobic

Biochem 2009;54:177–86.

amino acids. Secondly, replacement of the hydrophobic residues at

Kaiser H, Richter U, Keiner R, Brabant A, Hause B, Drager B, et al. Immunolocalisation

other sites except site S1 by polar, or basic or acidic amino acids of two tropinone reductases in potato (Solanum tuberosum L.) root, stolon, and

tuber sprouts. Planta 2006;225:127–37.

may block the tropinone reduction activity. This hypothesis is sup-

Keiner R, Kaiser H, Nakajima K, Hashimoto T, Dräger B. Molecular cloning, expression

ported by site-directed mutagenesis on DnTR2 that replacement of

and characterization of tropinone reductase II, an enzyme of the SDR family in

its residue Tyr-201 into Val at site S7 restored the tropinone reduc- Solanum tuberosum (L.). Plant Mol Biol 2002;48:299–308.

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the same protein fold. Proc Natl Acad Sci USA 1998;95:4876–81.

and 4-methylcyclohexanone, but cannot accept tropinone as its

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composite the substrate binding pocket. Finally, a site-directed

ical mobile signal for plant systemic acquired resistance. Science 2007;318:

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Acknowledgement stramonium root cultures by two speciﬁc reductases. Phytochemistry

1994;37:391–400.

Rothe G, Dräger B. Tropane alkaloids – metabolic response to carbohydrate signal in

This work is supported by the National Natural Science Foun-

root cultures of Atropa belladonna. Plant Sci 2002;163:979–85.

dation of China (No. 30870180, 31101417 and 31070298), the Schaller A, Stintzi A. Jasmonate biosynthesis and signaling for induced plant defense

against herbivory. In: Schaller A, editor. Induced plant resistance to herbivory.

Zhejiang Scientiﬁc and Technological Program (2008C12081), the

Dordrecht: Springer; 2008. p. 349–66.

Hangzhou Scientiﬁc and Technological Program (20080432T06,

Sharma M, Sharma A, Kumar A, Basu SK. Enhancement of secondary metabolites

20090233T15, 20101032B25) and Qianjiang Scholar Program. in cultured plant cells through stress stimulus. Am J Plant Physiol 2011;6:

50–71.

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