Abstract
Uridine diphosphate glycosyltransferases (UGTs) are pivotal in the process of glycosylation for decorating natural products with sugars. It is one of the versatile mechanisms in determining chemical complexity and diversity for the production of suite of pharmacologically active plant natural products. Picrorhiza kurrooa is a highly reputed medicinal herb known for its hepato-protective properties which are attributed to a novel group of iridoid glycosides known as picrosides. Although the plant is well studied in terms of its pharmacological properties, very little is known about the biosynthesis of these important secondary metabolites. In this study, we identified two family-1 glucosyltransferases from P. kurrooa. The full length cDNAs of UGT94F4 and UGT86C4 contained open reading frames of 1455 and 1422 nucleotides, encoding polypeptides of 484 and 473 amino acids respectively. UGT94F2 and UGT86C4 showed differential expression pattern in leaves, rhizomes and inflorescence. To elucidate whether the differential expression pattern of the two Picrorhiza UGTs correlate with transcriptional regulation via their promoters and to identify elements that could be recognized by known iridoid-specific transcription factors, upstream regions of each gene were isolated and scanned for putative cis-regulatory elements. Interestingly, the presence of cis-regulatory elements within the promoter regions of each gene correlated positively with their expression profiles in response to different phytohormones. HPLC analysis of picrosides extracted from different tissues and elicitor-treated samples showed a significant increase in picroside levels, corroborating well with the expression profile of UGT94F2 possibly indicating its implication in picroside biosynthesis. Using homology modeling and molecular docking studies, we provide an insight into the donor and acceptor specificities of both UGTs identified in this study. UGT94F2 was predicted to be an iridoid-specific glucosyltransferase having maximum binding affinity towards 7-deoxyloganetin while as UGT86C4 was predicted to be a kaempferol-specific glucosyltransferase. These are the first UGTs being reported from P. kurrooa.
Highlights
Glycosylation is a common modification reaction in plant metabolism and is invariably associated with the production of large array of secondary metabolites
To identify glucosyltransferases involved in the iridoid Oglucosylation from P. kurrooa, a homology based strategy was employed taking advantage of the highly conserved amino acid motif among plant secondary product glycosyltransferases (PSPG box) located in the C-terminal region
Understanding of various enzymatic or regulatory steps involved in secondary metabolite biosynthesis is a prerequisite for metabolic engineering
Summary
Glycosylation is a common modification reaction in plant metabolism and is invariably associated with the production of large array of secondary metabolites. The enzymes that lead to glycoside formation are known as uridine diphosphate glycosyltransferases (UGTs), members of family-1 glycosyltransferases superfamily. It contains over 80 families of enzymes [1,2] and typically accomplish this task by transferring a UDP-activated glucose to a corresponding acceptor molecule. Consistent with the variety and complexity of plant natural products, a large number of UGT gene sub-families have evolved for the glycosylation of these molecules [3,4]. UGTs involved in secondary metabolism share a conserved 44 amino acid residue motif (60–80% identity) known as the plant secondary product glucosyltransferase box (PSPG). UGTs share relatively low levels of sequence identity, especially within the regions related to acceptor binding, and this feature may be essential to account for the recognition of the huge variety of acceptors and the synthesis of the large number of products
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