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

• DOI: 10.1016/j.jbc.2021.101045
• Publication Date: 2021-08-04T22:37:43Z
• Authors: Payal Srivastava; Anchal Garg; Rajesh Chandra Misra; Chandan Singh Cha...; Sumit Ghosh
• Keywords: Labdane; Andrographolide; Andrographis Paniculata; Acanthaceae; Hydroxylation; Transferase

Glycosyltransferases constitute a large family of enzymes across all domains life, but knowledge their biochemical function remains largely incomplete, particularly in the context plant specialized metabolism. The labdane diterpenes represent class phytochemicals with many pharmacological benefits, such as anti-inflammatory, hepatoprotective, and anticarcinogenic. medicinal kalmegh (Andrographis paniculata) produces bioactive diterpenes; notably, C19-hydroxyl diterpene (andrograpanin) is predominantly found C19-O-glucoside (neoandrographolide), whereas having additional hydroxylation(s) at C3 (14-deoxy-11,12-didehydroandrographolide) or C14 (andrographolide) are primarily detected aglycones, signifying scaffold-selective C19-O-glucosylation planta. Here, we analyzed UDP-glycosyltransferase (UGT) activity levels various developmental stages tissues an apparent correlation UGT spatiotemporal accumulation neoandrographolide, major C19-O-glucoside. analysis recombinant UGTs preferentially expressed neoandrographolide-accumulating identified previously uncharacterized UGT86 member (ApUGT12/UGT86C11) that catalyzes strict scaffold selectivity. ApUGT12 localized to cytoplasm catalyzed substrate selectivity demonstrated by bacterium hosts was comparable native activity. Recombinant showed significantly higher catalytic efficiency using andrograpanin compared 14-deoxy-11,12-didehydroandrographolide trivial andrographolide. Moreover, silencing plants led drastic reduction neoandrographolide content increased andrograpanin. These data suggest involvement plants. This might help developing chemotypes synthesis pharmacologically relevant diterpenes. 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Furthermore, 23 screened assay, identification (UGT86C11) manner steady-state kinetic ApUGT12, altered profiles ApUGT12-silenced strong transcript expression patterns suggested pivotal understand diterpenes, conducted comprehensive profiling (andrographolide, 14-deoxy-11,12-didehydroandrographolide) C19-O-glucosides (neoandrographolide, andrographiside, 14-deoxy-11,12-didehydroandrographiside) five (germinating seeds, cotyledonary leaf stage, 15-day-old 30-day-old 60-day-old plants) six (root, leaf, stem, sepal, petal, seedpod) S1). HPLC methanolic extracts revealed andrographolide, leaves (Figs. 2A, S2, S3, B). these roots germinating seeds. addition, considerable amounts seedpod, stage seedlings, respectively. amount ready conversion C19-O-glucosylation, thus limiting 2A). On other hand, corresponding (andrographiside indicated inefficient results potentially distinct assays carried out total protein extract Andrograpanin, acceptors, served assays. Diterpene assay monitored HPLC. considerably followed seedpod 2B). contained seeds could neoandrographolide. Likewise, contrast, lower S4). achieved detectable level Thus, contributed rate andrographiside 14-deoxy-11,12-didehydroandrographiside biosynthesis, 2, B, Unlike (andrographolide bear hydroxyl group(s) positions. Therefore, strongly selective glucosides. Andrograpanin clear maximum noticed plants; however, roots. Similarly, RNA-Seq 170 million sequencing reads (41Garg transcripts encoding retrieved annotation database BlastX analysis. Among 615 annotated families, 161 categorized GT1/UGT family. Notably, UGT73AU1 (contig ApU2595) UGT5 ApU62177) S5A). previous study similar roots; different examined before know whether contribute determined quantitative RT-PCR (qRT-PCR) correlated S6A). sepal. substantially playing corroborate differential if considered S4) ApCPS2, initial pathway, hypothesized coincide 38 nonredundant S5A Table full-length coding sequences transcripts, assemblies generated studies consulted (https://medplantrnaseq.org/) (44Cherukupalli Divate Mittapelli S.R. Khareedu V.R.