Lin M,Yifeng Zhng,Wei Go,b,＊

a School of Traditional Chinese Medicine,Capital Medical University,Beijing 100069,China;

b Beijing Shijitan Hospital,Capital Medical University,Beijing 100038,China

Abstract 2-Oxoglutarate (2OG)-dependent dioxygenases (2-ODDs) are omnipresent iron-containing non-heme enzymes that catalyze various oxidation-reduction reactions in plant growth and development,nucleic acid modification and secondary metabolism.We systematically summarized recent research on the oxidative modifications of plant 2-ODDs and related enzymes,their vital importance in the biosynthesis of plant special metabolites,and their catalytic specificity/flexibility,and discussed the potential of 2-ODD as a new approach for the identification of pivotal genes and the elucidation of biosynthetic pathway.

Keywords:2-oxoglutarate-dependent dioxygenase;secondary metabolism;flavonoid;terpenoid;biosynthesis

1 Introduction

The 2-oxoglutarate-dependent dioxygenase(2-ODD) superfamily is the second largest enzyme family in the plant kingdom,and its members catalyze diversified oxidation-reduction reactions,including hydroxylation,epoxidation,halogenation,desaturation,isomerization,and ring expansion.

2-ODD is a soluble non-heme iron (II) enzyme found in the cytoplasmic matrix and is dependent on co-substrate 2-oxoglutarate (2OG),molecular oxygen,and cofactor iron (II).Compared with heme iron (II)oxidase such as cytochrome P450 (CYP450),2-ODD has more flexible metal coordination environment in which endogenous or exogenous ligands are more easily bound [1,2].The overall similarity of 2-ODD family is very low,and the conserved regions mainly include the characteristic double-stranded

β

-helix(DSBH) domain at the carboxyl terminal and the 2OG-FeII_Oxy motif formed by the coordination of 2OG,two histidine residues,one aspartic acid residue or glutamic acid residue to iron (II).

According to the similarity of amino acid sequence,Plant 2-ODD is divided into three categories:DOXA,DOXB and DOXC [3].DOXC is involved in the special metabolism of multiple metabolites.We constructed a phylogenetic tree of identified plant 2-ODDs amino acid sequences using neighbor joining method,all of which were clustered into DOXC branch (Fig.1).

Fig.1 Phylogenetic tree of identified plant 2-oxoglutarate-dependent dioxygenases

Using “plant,2-oxoglutarate-dependent dioxygenase,biosynthesis,and metabolism” as the keywords,we searched the literature related to plant 2-ODD in recent 20 years on

Pubmed,ScienceDirect and other websites.This review systematically summarized oxidative modifications of plant 2-ODD,its vital importance in the biosynthesis of plant special metabolites,and the research on catalytic specificity/flexibility,and discussed the potential of 2-ODD as a new approach for the identification of pivotal genes and the elucidation of the biosynthetic pathway.

2 2-ODDs involved in the metabolism of fatty acid derivatives

Plants contain many fatty acids and their derivatives,such as oleic acid,linoleic acid,salicylic acid and “stress hormone” jasmonic acid (JA) [4].Here JA is taken as an example to illustrate the importance of 2-ODD in the metabolism of fatty acid derivatives.

JA is an essential signal molecule in plant defense system [5].When faced with insect attack,pathogen infection or ultraviolet radiation,the increase of JA content activates the expression of defence genes and promotes the production of defensive substances in plants.However,the long-term maintenance of high-concentration of JA will inhibit plant growth.Therefore,plants need special mechanisms to inactivate JA in order to maintain a balance between plant defense and growth.Fig.2 shows the metabolic process associated with JA inactivation.

Fig.2 2-ODDs involved in the metabolism of fatty acid derivatives

2.1 JOXs/JAOs catalyzing the metabolic inactivation of jasmonic acid

The dicot

Arabidopsis thaliana

contains three special jasmonate-induced oxygenases (JOXs),which can directly catalyze the oxidation of JA to produce 12OH-JA

in vitro

[6].When JA was over-accumulated in the

JOXs

-RNAi plants,the expression level of defence genes was increased,the resistance to

Botrytis cinerea

and

Mamestra brassicae

was enhanced,and the growth of plant roots was inhibited.Another research group found three jasmonic acid oxidases (JAOs) that hydroxylate JA to 12OH-JA [7].

The simultaneous identification of JOXs/JAOs fills the gap in the JA metabolic pathway and is of great significance for the study of JA inactivation and subsequent JA-dependent defense downregulation.

2.2 Substrate specificity of JOXs/JAOs

The oxidation reaction mediated by JOXs/JAOs is substrate-specific and does not hydroxylate JA derivatives such as methyl jasmonate and JA-IIe.Liu’s team presented the X-ray crystal structure of the co-crystalline complex of

At

JOX2 with substrate JA,co-substrate 2OG and cofactor iron (II) and verified the substrate-specific binding mechanism of JOXs [8].

At

JOX2 is divided into three parts:the N-terminal region (M1-E218),the DSBH catalytic core (N219-P320) and the shorter C-terminal region(K321-R371).JA and 2OG exist in the cramped DSBH core,surrounded by

α

helices and loops on both sides.The position of JA in the DSBH core is strictly restricted by six hydrogen bonds and a Π accumulation residue F157,which defines the precise substrate localization and substrate specificity of JOXs/JAOs.

3 2-OD Ds involved in flavonoid metabolism

Flavonoids generally refer to the secondary metabolites of polyphenols with the C6-C3-C6 carbon skeleton formed by the interconnection of two benzene rings with phenolic hydroxyl groups (A-ring and B-ring) through three central carbon atoms.In plants,flavonoids are mainly biosynthesized through the shikimic acid pathway and subsequent phenylpropane metabolism [9].Under the action of chalcone isomerase (CHI),chalcone produced by chalcone synthase (CHS)undergoes intramolecular cyclization to give flavanones such as naringenin and eriodictyol.As the universal precursor of the flavonoid biosynthesis,flavanones can be catalyzed by myriad enzymes to generate flavonoids with various chemical structures and physiological activities.

The 2-ODD members involved in the flavonoid biosynthesis mainly include flavanone 3

β

-hydroxylase (F3H),flavonol synthase (FLS),type I flavone synthase (FNSI),and anthocyanidin synthase (ANS).In addition to the four typical 2-ODD members,there are many other 2-ODD enzymes along the metabolic pathway of flavonoids,such as the

Ob

F7ODM1 dioxygenase,which can catalyze the 7-O-demethylation of methoxylated flavones in

Ocimum basilicum

L [10].The role of the 2-ODD family in flavonoid biosynthesis is shown in Fig.3 with naringin as an example.

Fig.3 2-ODD family members involved in flavonoid metabolism

3.1 FNSI catalyzing the formation of flavones

Flavones are formed by the C-2,3 dehydrogenation of flavanones catalyzed by flavone synthase,in which FNSI is a 2-ODD dioxygenase and FNSⅡ is a CYP450 monooxygenase [9].Like

Zm

FNSI-1 and

At

DMR6,FNSI is concentrated in

Apiaceae

.Both

Zm

FNSI-1 and

At

DMR6 can import carbon-carbon double bonds at C-2,3 positions of naringin and eriodictyol to produce apigenin and luteolin [11].Additionally,the transgenic

Arabidopsis

expressing

ZmFNSI

and

ZmFNSII

can accumulate apigenin and protect themselves from ultraviolet radiation [12].FNSI enzyme in

Equisetum arvense

L was identified for the first time from the non-

Apiaceae

plant [13].FNSI from the liverwort

Plagiochasma appendiculatum

has the dual activity of FNSI/F2H and can convert naringenin into apigenin and 2-hydroxynaringenin [14].Compared with the angiosperm FNSI,the amino acid sequences of

Pa

FNSI/F2H and angiosperm F3H have higher homology,indicating the possible evolutionary relationship between liverwort FNSI and angiosperm F3H.In order to determine when and how F3H evolved,Cheng identified dozens of FNSI and F3H from plants of different evolutionary clades and found that the bifunctional enzymes FNSI/F2H in liverworts evolved into FNSI/F3H in

Physcomitrium patens

and

Selaginella moellendorffii

during the plant evolution process [15].FNSI/F3H weakened its FNSI activity in the

Gymnospermae

and finally evolved into F3H enzyme in the

Angiospermae

.This research provides the molecular basis for the chemical evolution of chalcones,flavanones,flavonoids to flavonols and anthocyanins in plant evolution,and will help plants adapt to harsh terrestrial ecosystem.

3.2 FLS catalyzing the formation of flavonols

Flavonol synthase (FLS) catalyzes the desaturation of C-2,3 position of flavanonols to form flavonols.Flavonol synthase encoded by

OsFLS

has the dual activity of FLS/F3H and tends to transform dihydrokaempferol rather than dihydroquercetin [16].The FLS from

Ornithogalum caudatum

was proved to have dual functions.In particular,its F3H activity did not require the presence of iron (II),but sufficient iron (II) supply was conducive to the continuous catalytic conversion of flavanonols to flavonols [17].

DoFLS1

from

Dendrobium officinale

was the first dioxygenase responsible for the formation of myricetin and would be up-regulated after the treatment with cold,drought or salicylic acid [18].

3.3 F3H catalyzing the formation of flavanonols

Flavanonols are compounds formed by introducing a hydroxyl group at the C-3 position of flavanones.F3H from

Artemisia annua

catalyzes naringin to generate dihydrokaempferol,producing flavonoids with both anti-malarial and anti-cancer effects [19].F3Hs catalyze naringin and regulate the early fiber development in two cotton species(

Gossypium hirsutum

and

G.barbadense

) [20].Overexpression of

OsF3H

in rice (

Oryza sativa

) can induce the expression of downstream genes related to anthocyanin and lead to the accumulation of kaempferol,quercetin and anthocyanin [21].

3.4 ANS catalyzing the formation of anthocyanidins

Anthocyanidin,also known as anthocyan,is a colored aglycone obtained by hydrolysis of anthocyanin.Dihydroflavonol 4-reductase (DFR)initiates the downstream pathway of anthocyanidin synthesis and selectively catalyzes the generation of corresponding leucoanthocyanidin from flavonols.Anthocyanidin synthase (ANS) can catalyze the conversion of leucoanthocyanidin into anthocyanidin in a red forma of

Perilla frutescens

[22].The same leucoanthocyanidin dioxygenase (LDOX)

was identified

from the

Arabidopsis

mutation

tds4-1

[23].Under the catalysis of glycosyltransferases,anthocyanidins can be converted into various anthocyanins to produce numerous colorful plants.Different from anthocyanins,proanthocyanidins(PAs) are polyphenolic polymers composed of flavanone subunits (-)-epicatechin and/or(+)-catechin [24].Due to the differences between the starter and extension subunits,there are multitudinous natural PAs with different chemical compositions and degrees of polymerization.Nevertheless,PAs consist almost exclusively of(-)-epicatechin subunits in the spornioderm of the model legume

Medicago truncatula

.Inconsistent with previous reports,the catalytic activities of

two LDOX/ANS

homologues are strikingly different in

M.truncatula

[25].Further studies demonstrated the existence of two independent parallel metabolic pathways for the synthesis of (-)-epicatechin starter and extension subunits in

M.truncatula

.In the LAR-independent pathway,ANS catalyzes leucoanthocyanidin to form anthocyanin,followed by ANR catalysis to form the(-)-epicatechin initial subunit.The LAR-dependent pathway (or LDOX pathway) starts from LAR and catalyzes the reduction of leucoanthocyanidin to form the (+)-catechin intermediate,which is then reduced by ANR to form the (-)-epicatechin extension subunit.Ultimately,the initial and extension subunits on two parallel pathways are polymerized to form PAs.The LDOX pathway explains the pattern of

LAR

gene in legumes,and its presence or absence results in the natural differences of PAs among different species.Efficient heterologous construction of biosynthetic pathway is important for the acquisition of valuable secondary metabolites.In order to obtain high-yield (+)-catechin,Zhao transformed the codonoptimized

CsF3H

,

AaDFR/CsDFR/FaDFR

and

DuLAR

high-copy expression vectors into

E.coli

Trans BL21(DE3),increased the copy number of

DFR/LAR

,the utilization of NADPH and oxygen,and finally obtained (+)-catechin (910.97 ±61.3 mg/L) from eriodictyol (1.0 g/L) in the optimized strain [26].

4 2-ODDs involved in steroidal glycoalkaloids metabolism

Steroidal glycoalkaloids (SGAs) in

Solanum

are compounds that have defensive effects on plant pathogens and predators,and some SGAs have anti-nutritional effects on humans.According to the structure of aglycone,SGAs are divided into solanidane-type and spirosolane-type.Representative solanidane-type SGAs include the toxic bitter glycoalkaloids

α

-solanine and

α

-chaconine in ulcerated,germinated or green potatoes.Representative spirosolane-type SGAs include

α

-tomatine in tomato leaves and immature fruits,

α

-solasonine and

α

-solamargine in eggplant,and

α

-solamarine and

β

-solamarine in wild-type potatoes.The biosynthesis of SGAs takes cholesterol as the precursor,and undergoes a series of C-16 oxidation,C-22 oxidation,C-26 oxidation and glycosylation reactions (Fig.4).Members of the 2-ODD family,termed GAME (GLYCOALKALOID METABOLISM),also participate in the metabolism of steroidal glycoalkaloids.

Fig.4 Four representative 2-ODDs involved in steroidal glycoalkaloids metabolism

4.1 GAME11 catalyzing the C-16 hydroxylation of hydroxycholesterol

Using CYP450 as the target,and through EST screening,cholesterol C-22 hydroxylase CYP72A188 (GAME7) and C-26 hydroxylase CYP72A208 (GAME8) were successfully detected in cultured potato (

Solanum tuberosum

),but the C-16 hydroxylase enzyme remained to be excavated [27].The following year,

St

16DOX (GAME11),the first 2-ODD enzyme involved in SGAs biosynthesis,was successfully discovered through deuteriumlabeled cholesterol feeding experiment.

St16DOX

is highly expressed in potato tuber buds with high accumulation of SGAs and its optimal substrate is(22S)-22,26-dihydroxycholesterol.SGAs content in

St16DOX

-RNAi plants was decreased,but the potato tuber yield was not affected.This phenomenon reveals the potential of

St

16DOX as a target gene to control the content of SGAs in potatoes and provides a new approach for cultivating low-content SGAs potatoes.

4.2 GAME31 catalyzing the C-23 hydroxylation of α-tomatine

Hydroxylation of

α

-tomatine is the first step to reduce bitterness and toxicity in the ripening of tomato fruit,and hydroxytomatine is the first compound along the pathway.The high expression of

GAME31

in ripe fruit is associated with the content of hydroxytomatine during fruit development of cultivated and wild-type tomatoes (

S.pennellii

) [28].Recombinant GAME31 enzyme assays verified the conversion of

α

-tomatine to hydroxytomatine in the reaction system containing GAME31,2OG,ascorbic acid and iron (II).In particular,GAME31 could only catalyze the spirosolane-type SGAs in tomato and eggplant,but not the solanidane-type SGAs in potato.Another research team from Kobe University in Japan,who had previously successfully excavated C-16/22/26 hydroxylase,also found C-23

α

hydroxylase

Sl

23DOX from cultivated tomato (

S.lycopersicum

),the catalytic product of which could spontaneously isomerize to form neorickiioside B(also named hydroxytomatine) [29].Lycoperoside C(23-O-acetylated neorickiioside B) was also detected in the leaves of

Sl23DOX

-overexpressed tomato plants.

4.3 GAME32 catalyzing the biosynthesis of leptinine

In addition to GAME31,Asaph Aharoni’s research group also used leptinine,a defensive substance unique to wild-type potato (

S.chacoense

),to carry out the study on the solanidane-type SGAs hydroxylase [28].They obtained two related GAME32 proteins,

Sc

GAME32-M6 and

Sc

GAME32-like-M6,based on the specific accumulation pattern of leptinine in leaves,stems and green flowers.Prokaryotic expression and

in vitro

enzymatic reaction showed that

Sc

GAME32-M6 was involved in the key intermediate step of leptinine biosynthesis and specifically catalyzed the hydroxylation of

α

-chaconine and

α

-solanine to form leptinine I and leptinine II,enabling the plants to resist the invasion of Colorado Potato Beetle(

Leptinotarsa decemlineata

Say).

4.4 DPS catalyzing the rearrangement of spirosolane to form solanidane through C-16 hydroxylation

As previously mentioned,some wild-type potatoes contain

α

-solamarine and

β

-solamarine(spirosolane-type SGAs) [30],while most cultivated potatoes only contain

α

-solanine and

α

-chaconine(solanidane-type SGAs),whose glycosyl moieties are identical with

α

-solamarine and

β

-solamarine [31].Researchers observed that

α

-solanine/

α

-chaconine was restored when

α

-solamarine/

β

-solamarine or their aglycones was fed to the hairy roots of SGAsdeficient potatos,which verified the conjecture that the modification of spirosolane-type skeleton in potato would lead to the formation of solanidanetype skeleton [32].In conclusion,DPS (Dioxygenase for Potato Solanidane synthesis) catalyzes the rearrangement of spirosolane to form solanidane through C-16 hydroxylation.Compared with the metabolic detoxification of

Sl

23DOX/GAME31,the DPS-catalyzed conversion to toxic SGAs represents the biological evolutionary advantage,allowing plants to produce more toxic and defensive SGAs to avoid devastating disasters caused by invaders.

5 2-ODDs involved in terpenoid metabolism

Terpenoids,widely distributed in plants,mainly include phytohormones (gibberellins and abscisic acid) and secondary metabolites with special pharmacological activities,such as taxol [33],artemisinin [34] and gossypol [35].Fig.5 illustrates the significance of 2-ODDs in terpenoid metabolism with gibberellin as an example.

Fig.5 2-ODD family members involved in gibberellin metabolism

Gibberellin (GA) is a tetracyclic diterpenoid phytohormone with gibbane as the skeleton,and its regulatory effect runs through the whole life cycle.2-ODD family members,such as GA20-oxidase(GA20ox),GA3-oxidase (GA3ox),GA2-oxidase(GA2ox) and other special gibberellin oxidases,are mainly involved in the downstream biosynthetic pathway of GA.

5.1 GA20ox catalyzing the formation of bioactive GA precursors

Similar to other plants,

Arabidopsis

GA20ox can oxidate GAat C-20 to produce a bioactive GA precursor GA[36].GA20ox plays a considerable role in the regulation of asexual and sexual reproduction in plants.In woodland diploid strawberry

(

Fragaria vesca

),

FvGA20ox4

expressed in the axillary meristem and developmental stolon is responsible for the formation of strawberry stolons and the maintenance of the balance between sexual reproduction and asexual reproduction [37].In some self-incompatible plants such as pears(

Pyrus bretschneideri

Rehd.),another valuable trait is parthenocarpy.Overexpression of

PbGA20ox2

could enhance the synthesis of GA,producing the seedless fruit of stamen-removal flowers without fertilization [38].2,4-dichlorophenoxyacetic acid treatment could up-regulate the expression levels of

PbGA20ox2-like

and

PbGA3ox-1

,thus enhancing the biosynthesis of GAand promoting the development of parthenocarpy [39].

5.2 GA3ox catalyzing the formation of bioactive GA

GA3ox participates in the downstream catalytic reaction of GA20ox and can directly catalyze GAto synthesize bioactive GA.

Cs

GA5ox of cucumber(

Cucumis sativus

) catalyzes the C-3 oxidation of GAto produce GA[40].Whereas,

Cs

GA1ox/ds,lacking C-3 oxidation activity,can catalyze the C-1

β

oxidation of GAto produce GAand the desaturation of C-9,11 of GAto produce GA.After replacing the three amino acids Phe93,Pro106 and Ser202 of

Cs

GA1ox/ds with the typical conserved amino acids Tyr93,Met106 and Thr202 of GA3ox,

Cs

GA1ox/ds restored gibberellin 3-oxidase activity.Meanwhile,mutation of the corresponding sites in

Cs

GA3ox5 may cause the loss of gibberellin C-3 oxidase activity,

indicating the importance of these three conserved sites in maintaining gibberellin C-3 oxidase activity

.

The function loss of

Cl

GA3ox causes dwarf characteristic in watermelon (

Citrullus lanatus

L.) [41].Specifically,the gene

Cla015407

encoding GA3ox in dwarf watermelon has a SNP mutation (G→A) at the intron splice acceptor site,which causes protein truncation and loss of functional 2OG-FeII_Oxy domain.Consequently,the expression of

Cla015407

and other genes related to GA synthesis is downregulated and the content of GAis significantly decreased,resulting in a dwarf phenotype.

5.3 GA2ox catalyzing the deactivation of bioactive GA

GA2ox inactivated the biologically active GA or its precursors via 2

β

-hydroxylation reaction.

Pc

GA2ox1 from runner bean

(

Phaseolus coccineus

L.)was the first identified gibberellin C-2 oxidase which catalyzed continuous C-2 oxidation of GAand GAto form GA,GAand the catabolites yielded from GAor GAby further oxidation at C-2 [42].

At

GA2ox1/2/3 in

Arabidopsis

also encoded functional gibberellin C-2 oxidases [42].In addition,transgenic switchgrass (

Panicum virgatum

L.)overexpressing

PvGA2ox5

and

PvGA2ox9

exhibited a special GA-deficient phenotype,including dark green leaves,semi-dwarf,increased tiller and reduced lignin content [43].

5.4 Other functional gibberellin oxidases

Expect for GA20ox,GA3ox and GA2ox,there are many other 2-ODDs in plants which catalyze the hydroxylation of other sites in plants.

Cs

GA7ox1/2 in Cucumber catalyzes the carboxylation of aldehyde-GAto produce GA,and

Cs

GA7ox1 catalyzes GAto form multiple products (GA,15

α

-OH GAand 15

α

-OH GA).Notably,

Cs

GA7ox2 catalyzes GAto form 12

α

-OH analogue GA[44].

At

GAS2 (Gain of Function in ABA-modulated Seed Germination 2) catalyzes the directional hydration of GAat C-16,17 to generate bioactive 16

α

-OH GA(DHGA) [45].In

Tripterygium wilfordii

,the protein encoded by

TwGA13ox

catalyzes the C-13 hydroxylation of GAto form GA[46].

TaGA1ox-B1

and

HvGA3

,

18ox1

from

wheat (

Triticum aestivum

L.) and barley(

Hordeum vulgare

L

.

) catalyze the production of a large amount of 1

β

-OH GAs and rare 18-OH GAs [47].

6 Conclusion

Most of the secondary metabolites of medicinal plants have good protective effects on the plants themselves and also have important pharmacological activities.At present,many new drugs modified according to the structure of natural products have entered the clinical trial phase.Thus,we need a richer source of natural products to meet the needs of drug development.Most natural products have complex structures and are difficult to be synthesized by chemical methods.Meanwhile,some plants have long growth cycle and little accumulation of bioactive ingredients.Therefore,total chemical synthesis and traditional plant extraction methods can not meet the growing market need.The development of molecular biology and synthetic biology has brought new impetus to the acquisition of bioactive ingredients.By analyzing the plant-derived biosynthetic pathway of bioactive ingredients,the artificially-designed pathways have been loaded into engineered bacteria such as

Escherichia coli

to construct cell factories and realize the efficient heterologous production of bioactive ingredients or their precursors.

2-ODD,the second largest family in the plant genome,is involved in the biosynthesis of various crucial substances.Current 2-ODD research focuses on the regulation of plant growth and development.The development of genomics,transcriptomics,metabolomics has greatly increased the substrate spectrum and catalysis type of 2-ODD,providing new ideas for further research on the biosynthesis of active ingredients in natural medicinal plants.In order to reduce the waste of plant resources,the formation mechanism of the active ingredients needs to be further studied.With the joint efforts of researchers from all over the world,more and more biosynthesis-related genetic elements will be excavated to reach the goal of industrial production of important active ingredients,thus alleviating the shortage of resources and realizing the sustainable and high-quality development of traditional Chinese medicine.

Acknowledgments

This work was supported by the National Key R&D Program of China (2020YFA0908000),the National Natural Science Foundation of China(81773830),the Key Project at central government level:The ability establishment of sustainable use for valuable Chinese medicine resources (2060302-1806-03),and National Program for Special Support of Eminent Professionals.