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UGT86C11 is a novel plant UDP-glycosyltransferase involved in labdane diterpene biosynthesis

2021; Elsevier BV; Volume: 297; Issue: 3 Linguagem: Inglês

10.1016/j.jbc.2021.101045

1083-351X

Payal Srivastava, Anchal Garg, Rajesh Chandra Misra, Chandan Singh Chanotiya, Sumit Ghosh,

Toxin Mechanisms and Immunotoxins

Glycosyltransferases constitute a large family of enzymes across all domains of life, but knowledge of their biochemical function remains largely incomplete, particularly in the context of plant specialized metabolism. The labdane diterpenes represent a large class of phytochemicals with many pharmacological benefits, such as anti-inflammatory, hepatoprotective, and anticarcinogenic. The medicinal plant kalmegh (Andrographis paniculata) produces bioactive labdane diterpenes; notably, the C19-hydroxyl diterpene (andrograpanin) is predominantly found as C19-O-glucoside (neoandrographolide), whereas diterpenes having additional hydroxylation(s) at C3 (14-deoxy-11,12-didehydroandrographolide) or C3 and C14 (andrographolide) are primarily detected as aglycones, signifying scaffold-selective C19-O-glucosylation of diterpenes in planta. Here, we analyzed UDP-glycosyltransferase (UGT) activity and diterpene levels across various developmental stages and tissues and found an apparent correlation of UGT activity with the spatiotemporal accumulation of neoandrographolide, the major diterpene C19-O-glucoside. The biochemical analysis of recombinant UGTs preferentially expressed in neoandrographolide-accumulating tissues identified a previously uncharacterized UGT86 member (ApUGT12/UGT86C11) that catalyzes C19-O-glucosylation of diterpenes with strict scaffold selectivity. ApUGT12 localized to the cytoplasm and catalyzed diterpene C19-O-glucosylation in planta. The substrate selectivity demonstrated by the recombinant ApUGT12 expressed in plant and bacterium hosts was comparable to native UGT activity. Recombinant ApUGT12 showed significantly higher catalytic efficiency using andrograpanin compared with 14-deoxy-11,12-didehydroandrographolide and trivial activity using andrographolide. Moreover, ApUGT12 silencing in plants led to a drastic reduction in neoandrographolide content and increased levels of andrograpanin. These data suggest the involvement of ApUGT12 in scaffold-selective C19-O-glucosylation of labdane diterpenes in plants. This knowledge of UGT86 function might help in developing plant chemotypes and synthesis of pharmacologically relevant diterpenes. Glycosyltransferases constitute a large family of enzymes across all domains of life, but knowledge of their biochemical function remains largely incomplete, particularly in the context of plant specialized metabolism. The labdane diterpenes represent a large class of phytochemicals with many pharmacological benefits, such as anti-inflammatory, hepatoprotective, and anticarcinogenic. The medicinal plant kalmegh (Andrographis paniculata) produces bioactive labdane diterpenes; notably, the C19-hydroxyl diterpene (andrograpanin) is predominantly found as C19-O-glucoside (neoandrographolide), whereas diterpenes having additional hydroxylation(s) at C3 (14-deoxy-11,12-didehydroandrographolide) or C3 and C14 (andrographolide) are primarily detected as aglycones, signifying scaffold-selective C19-O-glucosylation of diterpenes in planta. Here, we analyzed UDP-glycosyltransferase (UGT) activity and diterpene levels across various developmental stages and tissues and found an apparent correlation of UGT activity with the spatiotemporal accumulation of neoandrographolide, the major diterpene C19-O-glucoside. The biochemical analysis of recombinant UGTs preferentially expressed in neoandrographolide-accumulating tissues identified a previously uncharacterized UGT86 member (ApUGT12/UGT86C11) that catalyzes C19-O-glucosylation of diterpenes with strict scaffold selectivity. ApUGT12 localized to the cytoplasm and catalyzed diterpene C19-O-glucosylation in planta. The substrate selectivity demonstrated by the recombinant ApUGT12 expressed in plant and bacterium hosts was comparable to native UGT activity. Recombinant ApUGT12 showed significantly higher catalytic efficiency using andrograpanin compared with 14-deoxy-11,12-didehydroandrographolide and trivial activity using andrographolide. Moreover, ApUGT12 silencing in plants led to a drastic reduction in neoandrographolide content and increased levels of andrograpanin. These data suggest the involvement of ApUGT12 in scaffold-selective C19-O-glucosylation of labdane diterpenes in plants. This knowledge of UGT86 function might help in developing plant chemotypes and synthesis of pharmacologically relevant diterpenes. Plants make an astonishing diversity of chemicals that are important to their overall fitness in a challenging environment and are also valuable as pharmaceuticals and various industrial chemicals (1Pichersky E. Raguso R.A. Why do plants produce so many terpenoid compounds?.New Phytol. 2018; 220: 692-702Crossref PubMed Scopus (182) Google Scholar, 2Lacchini E. Goossens A. Combinatorial control of plant specialized metabolism: Mechanisms, functions, and consequences.Annu. Rev. Cell. Dev. Biol. 2020; 36: 291-313Crossref PubMed Scopus (15) Google Scholar, 3Erb M. Kliebenstein D.J. Plant secondary metabolites as defenses, regulators, and primary metabolites: The blurred functional trichotomy.Plant Physiol. 2020; 184: 39-52Crossref PubMed Google Scholar). The diversity in plant specialized pathway is believed to have originated because of the evolution of diverse enzyme function following gene duplication and neofunctionalization (4Fernie A.R. Tohge T. 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Sharma S. Ghosh S. Andrographis paniculata transcriptome provides molecular insights into tissue-specific accumulation of medicinal diterpenes.BMC Genomics. 2015; 16: 659Crossref PubMed Scopus (53) Google Scholar). The final step in neoandrographolide biosynthesis involves C19-O-glucosylation of andrograpanin (Fig. 1). However, UGT involved in developmental and tissue-specific biosynthesis of neoandrographolide was not identified. Previously, two methyl jasmonate (MeJA)–inducible UGTs (UGT73AU1 and UGT5), which catalyzed in vitro C19-O-glucosylation of andrograpanin, were identified, but their involvement in planta biosynthesis of neoandrographolide was not understood (42Li Y. Lin H.X. Wang J. Yang J. Lai C.J. Wang X. Ma B.W. Tang J.F. Li Y. Li X.L. Guo J. Gao W. Huang L.Q. Glucosyltransferase capable of catalyzing the last step in neoandrographolide biosynthesis.Org. Lett. 2018; 20: 5999-6002Crossref PubMed Scopus (11) Google Scholar, 43Sun W. Leng L. Yin Q. Xu M. Huang M. Xu Z. Zhang Y. Yao H. Wang C. Xiong C. Chen S. Jiang C. Xie N. Zheng X. Wang Y. et al.The genome of the medicinal plant Andrographis paniculata provides insight into the biosynthesis of the bioactive diterpenoid neoandrographolide.Plant J. 2019; 97: 841-857Crossref PubMed Scopus (44) Google Scholar). To investigate the UGT involved in spatiotemporal biosynthesis of neoandrographolide, we have analyzed a large-scale transcriptome data of kalmegh and identified UGTs that preferentially expressed in neoandrographolide-accumulating tissues. Furthermore, 23 recombinant UGTs were screened in UGT assay, leading to the identification of ApUGT12 (UGT86C11) catalyzing C19-O-glucosylation of diterpenes in a scaffold-selective manner (Fig. 1). The steady-state kinetic of ApUGT12, an altered diterpene profiles in ApUGT12-silenced plants, and a strong correlation of ApUGT12 transcript expression with UGT activity and neoandrographolide accumulation patterns across various developmental stages and tissues suggested a pivotal role of ApUGT12 in the biosynthesis of diterpene C19-O-glucoside. To understand spatiotemporal biosynthesis of diterpenes, we conducted comprehensive profiling of diterpene aglycones (andrographolide, andrograpanin, and 14-deoxy-11,12-didehydroandrographolide) and diterpene C19-O-glucosides (neoandrographolide, andrographiside, and 14-deoxy-11,12-didehydroandrographiside) across five developmental stages (germinating seeds, cotyledonary leaf stage, 15-day-old plants, 30-day-old plants, and 60-day-old plants) using six tissues (root, leaf, stem, sepal, petal, and seedpod) (Fig. S1). HPLC analysis of methanolic extracts revealed higher content of neoandrographolide, andrographolide, and 14-deoxy-11,12-didehydroandrographolide in leaves (Figs. 2A, S2, and S3, A and B). However, these diterpenes were not detected in roots and germinating seeds. In addition, neoandrographolide, andrographolide, and 14-deoxy-11,12-didehydroandrographolide were also detected in considerable amounts in seedpod, sepal, and cotyledonary leaf stage seedlings, respectively. The increased amount of neoandrographolide than andrograpanin in kalmegh tissues suggested ready conversion of andrograpanin to neoandrographolide following C19-O-glucosylation, thus limiting in planta accumulation of andrograpanin (Figs. 1 and 2A). On the other hand, higher content of andrographolide and 14-deoxy-11,12-didehydroandrographolide than the corresponding C19-O-glucosides (andrographiside and 14-deoxy-11,12-didehydroandrographiside) indicated inefficient C19-O-glucosylation of andrographolide and 14-deoxy-11,12-didehydroandrographolide (Figs. 1 and S3, A and B). These results suggest that scaffold-selective C19-O-glucosylation potentially contributes to distinct patterns of diterpene aglycones and glucosides in planta. To investigate the involvement of UGT in diterpene C19-O-glucosylation, UGT assays were carried out using total protein extract of various tissues. Andrograpanin, andrographolide, and 14-deoxy-11,12-didehydroandrographolide were the sugar acceptors, whereas UDP-glucose served as sugar donor in assays. Diterpene C19-O-glucoside produced in assay was monitored by HPLC. UGT assays using andrograpanin as sugar acceptor revealed considerably higher enzyme activity in leaves of 60-day-old and 30-day-old plants followed by in leaves of 15-day-old plants and seedpod (Fig. 2B). These tissues also contained higher amount of neoandrographolide (Fig. 2A). UGT assay using total protein extract of roots and germinating seeds could not form neoandrographolide. Likewise, neoandrographolide was not detected in roots and germinating seeds. In contrast, UGT assay using 14-deoxy-11,12-didehydroandrographolide revealed considerably lower activity than using andrograpanin (Fig. S4). However, C19-O-glucosylation of andrographolide could not be achieved at a detectable level using total protein extract of various tissues. Thus, higher UGT activity using andrograpanin than andrographolide and 14-deoxy-11,12-didehydroandrographolide might have contributed to neoandrographolide biosynthesis at a higher rate than andrographiside and 14-deoxy-11,12-didehydroandrographiside biosynthesis, leading to distinct profiles of diterpene aglycones and C19-O-glucosides in planta (Figs. 2, A and B, S3, A and B, and S4). Unlike andrograpanin, the other two diterpene aglycones (andrographolide and 14-deoxy-11,12-didehydroandrographolide) bear additional hydroxyl group(s) at the C3 and/or C14 positions. Therefore, these results strongly suggest that scaffold-selective C19-O-glucosylation of diterpene aglycones by UGT potentially contributes to selective accumulation of diterpene aglycones and glucosides. Andrograpanin C19-O-glucosylation activity and neoandrographolide level in various tissues showed a clear correlation (Fig. 2, A and B). The maximum UGT activity toward andrograpanin C19-O-glucosylation was noticed in leaves of 60-day-old plants; however, UGT activity was not detected in roots. Similarly, neoandrographolide was detected at a higher level in leaves of 60-day-old plants, whereas neoandrographolide could not be detected in roots. To investigate the enzyme involved in C19-O-glucosylation of diterpenes, a large-scale RNA-Seq data representing more than 170 million sequencing reads of leaves and roots were screened and UGTs that preferentially expressed in leaves were identified (41Garg A. Agrawal L. Misra R.C. Sharma S. Ghosh S. Andrographis paniculata transcriptome provides molecular insights into tissue-specific accumulation of medicinal diterpenes.BMC Genomics. 2015; 16: 659Crossref PubMed Scopus (53) Google Scholar). The transcripts potentially encoding UGTs were retrieved based on annotation to the Carbohydrate-Active enZYymes database following BlastX analysis. Among a total of 615 transcripts annotated to various GT families, 161 transcripts were categorized under the GT1/UGT family. Notably, UGT73AU1 (contig ApU2595) and UGT5 (contig ApU62177) transcript expression in leaves and roots was quite comparable (Fig. S5A). The previous study also found a similar transcript expression of UGT73AU1 in leaves and roots; however, UGT5 transcript expression in different tissues was not examined before (42Li Y. Lin H.X. Wang J. Yang J. Lai C.J. Wang X. Ma B.W. Tang J.F. Li Y. Li X.L. Guo J. Gao W. Huang L.Q. Glucosyltransferase capable of catalyzing the last step in neoandrographolide biosynthesis.Org. Lett. 2018; 20: 5999-6002Crossref PubMed Scopus (11) Google Scholar, 43Sun W. Leng L. Yin Q. Xu M. Huang M. Xu Z. Zhang Y. Yao H. Wang C. Xiong C. Chen S. Jiang C. Xie N. Zheng X. Wang Y. et al.The genome of the medicinal plant Andrographis paniculata provides insight into the biosynthesis of the bioactive diterpenoid neoandrographolide.Plant J. 2019; 97: 841-857Crossref PubMed Scopus (44) Google Scholar). To know whether UGT73AU1 and UGT5 contribute to developmental and tissue-specific C19-O-glucosylation of diterpenes, UGT73AU1 and UGT5 transcript expression was determined by quantitative RT-PCR (qRT-PCR) analysis and correlated with diterpene accumulation patterns and UGT activity (Fig. S6A). UGT73AU1 and UGT5 transcripts expressed at a higher level in roots of 60-day-old plants and sepal. However, andrograpanin C19-O-glucosylation activity and neoandrographolide content in these tissues were substantially lower or not detected (Fig. 2, A and B). Therefore, UGT73AU1 and UGT5 transcript expression patterns strongly indicated that they might not be playing a major role in developmental and tissue-specific biosynthesis of diterpene glucosides. Moreover, a similar catalytic efficiency of recombinant UGT5 using andrograpanin, andrographolide, and 14-deoxy-11,12-didehydroandrographolide could not corroborate differential activity of native UGT using these diterpene aglycones, if UGT5 is considered to be playing a major role in planta diterpene C19-O-glucosylation (Fig. S4) (42Li Y. Lin H.X. Wang J. Yang J. Lai C.J. Wang X. Ma B.W. Tang J.F. Li Y. Li X.L. Guo J. Gao W. Huang L.Q. Glucosyltransferase capable of catalyzing the last step in neoandrographolide biosynthesis.Org. Lett. 2018; 20: 5999-6002Crossref PubMed Scopus (11) Google Scholar). The transcript expression of ApCPS2, which catalyzed the initial diterpene cyclization reaction in the neoandrographolide biosynthetic pathway, showed a strong correlation with developmental and tissue-specific biosynthesis of neoandrographolide (40Misra R.C. Garg A. Roy S. Chanotiya C.S. Vasudev P.G. Ghosh S. Involvement of an ent-copalyl diphosphate synthase in tissue-specific accumulation of specialized diterpenes in Andrographis paniculata.Plant Sci. 2015; 240: 50-64Crossref PubMed Scopus (20) Google Scholar, 41Garg A. Agrawal L. Misra R.C. Sharma S. Ghosh S. Andrographis paniculata transcriptome provides molecular insights into tissue-specific accumulation of medicinal diterpenes.BMC Genomics. 2015; 16: 659Crossref PubMed Scopus (53) Google Scholar). Therefore, it could be hypothesized that UGT transcript expression might also coincide with in planta biosynthesis of neoandrographolide. The analysis of RNA-Seq data identified 38 nonredundant UGT transcripts that expressed at a higher level in neoandrographolide-accumulating leaves than in roots (Fig. S5A and Table S1). To extract full-length coding sequences of the transcripts, transcriptome assemblies generated in other studies were also consulted (https://medplantrnaseq.org/) (44Cherukupalli N. Divate M. Mittapelli S.R. Khareedu V.R.