Probing the Single Key Amino Acid Responsible for the Novel Catalytic Function of ent-Kaurene Oxidase Supported by NADPH-Cytochrome P450 Reductases in Tripterygium wilfordii.

Ping Su,Tianyuan Hu,Yujun Zhao,Yuru Tong,Wei Gao,Baowei Ma,Hongyu Guan,Yifeng Zhang,Lichan Tu,Linhui Gao,Xing Wang,Luqi Huang,Jiawei Zhou

doi:10.3389/fpls.2017.01756

Abstract

Tripterygium wilfordii produces not only ent-kaurene, which is an intermediate of gibberellin (GA) biosynthesis in flowering plants, but also 16α-hydroxy-ent-kaurane, whose physiological role has not been characterized. The two compounds are biosynthesized from the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) by diterpene synthases, which have been discovered and functionally characterized in T. wilfordii. Here, we described the functional characterization of four cytochrome P450 reductases (TwCPR) and one ent-kaurene oxidase (TwKO). Four TwCPRs were found to have relatively ubiquitous expression in T. wilfordii root, stem, leaf, and flower tissues. Co-expression of both a TwCPR and TwKO in yeast showed that TwCPR3 has a slightly better activity for providing the electrons required for these reactions, indicating that TwCPR3 is more suitable for use in the functional analysis of other cytochrome P450 monooxygenases. TwKO catalyzed the three-step oxidation of the C4α methyl of the tetracyclic diterpene intermediate ent-kaurene to form ent-kaurenoic acid as an early step in GA biosynthesis. Notably, TwKO could also convert 16α-hydroxy-ent-kaurane to 16α-hydroxy-ent-kaurenoic acid, indicating an important function of 16α-hydroxy-ent-kaurane in the anti-HIV principle tripterifordin biosynthetic pathway in planta. Homology modeling and molecular docking were used to investigate the unknown crucial active amino acid residue involved in the catalytic reaction of TwKO, and one key residue (Leu387) contributed to the formation of 16α-hydroxy-ent-kaurenoic acid, most likely by forming hydrogen bonds with the hydroxyl group (-OH) of 16α-hydroxy-ent-kaurane, which laid the basis for further investigation of the multifunctional nature of KO catalysis. Also, our findings paved the way for the complete biosynthesis of the anti-HIV principle tripterifordin.

Highlights

Tripterygium wilfordii Hook. f. has a long history of use in traditional Chinese medicine, mainly to treat rheumatoid arthritis (Tu, 2009)
More than 20 labdane-related diterpenoid (LRD) olefins were used to thoroughly investigate the enzymatic promiscuity, and the results showed OsKO2 was much more specific (specific for ent-(iso)kaurene and the Abbreviations: CPR, cytochrome P450 reductase; CTAB, cetyltrimethylammonium bromide; 2,4-D, 2,4-dichlorophenoxyacetic acid; DCPIP, dichlorophenolindophenol; ent-CPP, ent-copalyl diphosphate; GAs, gibberellins; gas chromatography–mass spectrometry (GC–MS), gas chromatography–mass spectrometer; GGPP, geranylgeranyl diphosphate; GO, Gene Ontology database; indole3-butyric acid (IBA), indole-3-butyric acid; KO, ent-kaurene oxidase; KOG/COG, Clusters of Orthologous Groups of proteins; KT, kinetin; LB, Luria-Bertani; LRD, labdane-related diterpenoid; MBP, maltosebinding protein; MS, Murashige and Skoog; Nr, NCBI non-redundant protein sequences; Nt, NCBI non-redundant nucleotide sequences; ORF, open reading frame; Pfam, Protein family; Swiss-Prot, a manually annotated and reviewed protein sequence database
Tripterygium wilfordii suspension cells were cultured in Murashige and Skoog (MS) medium containing 30 g/L sucrose supplemented with 0.1 mg/L kinetin (KT), 0.5 mg/L indole3-butyric acid (IBA), and 0.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and maintained at 25 ± 1◦C while being shaken at 120 rpm in the dark (Su et al, 2014)

Summary

INTRODUCTION

Tripterygium wilfordii Hook. f. has a long history of use in traditional Chinese medicine, mainly to treat rheumatoid arthritis (Tu, 2009). Ent-kaurene oxidase (KO) from the CYP701 family, has been reported to catalyze the three-step oxidation of the C4α methyl of the tetracyclic diterpene intermediate entkaurene to form ent-kaurenoic acid as an early step in GA biosynthesis (Morrone et al, 2010; Hedden and Thomas, 2012) Such catalytic activities strictly depend on their partner, NADPHcytochrome P450 reductase (CPR), to provide the necessary electrons. Structurally closely related ent-trachylobane), whereas AtKO was found to exhibit much broader promiscuity, targeting the C4α methyl of ent-CPP-derived LRDs and the C2α and/or C3β of normal and syn-CPP-derived LRDs to produce hydroxylated or carboxylated derivatives (Mafu et al, 2016) These results prompted us to explore the promiscuity of TwKO and to investigate whether it reacted with ent-kaurene as well as 16α-hydroxy-ent-kaurane in T. wilfordii. The forming of 16α-hydroxy-entkaurenoic acid catalyzed by TwKO, paved the way for the complete biosynthesis of the tripterifordin and provided the precursor for biosynthesis of other bioactive diterpenoids, e.g., ent-16α, 17-dihydroxy-kauran-19-oic acid (Chen et al, 1992; Sung et al, 2011)

MATERIALS AND METHODS

RESULTS

DISCUSSION