Enzymatic O-Glycosylation of Etoposide Aglycone by Exploration of the Substrate Promiscuity for Glycosyltransferases

Abstract

The 4-O-β-D-glucopyranoside of DMEP ((−)-4′-desmethylepipodophyllotoxin) (GDMEP), a natural product from Podophyllum hexandrum, is the direct precursor to the topoisomerase inhibitor etoposide, used in dozens of chemotherapy regimens for various malignancies. The biosynthesis pathway for DMEP has been completed, while the enzyme for biosynthesizing GDMEP is still unclear. Here, we report the enzymatic O-glycosylation of DMEP with 53% conversion by exploring the substrate promiscuity and entrances of glycosyltransferases. Notably, we found 6 essential amino acid residues surrounding the putative substrate entrances exposed to the protein surface in UGT78D2, CsUGT78D2, and CsUGT78D2-like, and these residues may determine substrate specificity and high O-glycosylation activity toward DMEP. Our results provide an effective route for one-step synthesis of GDMEP. Identification of the key residues and entrances of glycosyltransferases will promote precise identification of glycosyltransferase biocatalysts for novel substrates and provide a rational basis for glycosyltransferase engineering.

Biosynthesis, total synthesis, and pharmacological activities of aryltetralin-type lignan podophyllotoxin and its derivatives

“…Natural Product Reports (À)-PTOX (Scheme 4). Researchers rst used the oxidative enolate coupling methodology to couple oxazolidinone (30) with an ester (31) to form a single diastereomer dibenzylbutyrolactone (32) as the natural substrate of the DPS enzyme. Then, they used chaperones GroEL and GroES to optimize the soluble expression level of the DPS enzyme, leading 32 to completely transform into (À)-DPS (19).…”

“…[26][27][28] Therefore, the key to realizing the heterologous biosynthesis of PTOX is to identify its biosynthetic pathway genes. By 2019, the biosynthetic pathway of PTOX and its derivatives have almost been revealed, 29,30 making it possible for heterologous biosynthesis of pathway intermediates. For example, using the latestage biosynthetic precursor DPT has realized milligram-scale production, both in Escherichia coli and plant classis.…”

YALI0C22088g from Yarrowia lipolytica catalyses the conversion of l‐methionine into volatile organic sulfur‐containing compounds

“…To locate the key amino acid residues that determine the high L ‐ Met depletion activity of YALI0C22088g, we compared the amino acid sequence of YALI0C22088g to those of YHR112C and KLLA0_E21319g, which share 54.40% and 54.79% of their identities with YALI0C22088g, respectively, in according with the methods described by Jia et␣al. (2019). Twelve out of 29 amino acid residues constituting the entrance, tunnel and active site in YALI0C22088g were different from those in YHR112C and KLLA0_E21319g (Fig.…”

Promiscuity and specificity of eukaryotic glycosyltransferases

“…See the text for details of each reaction. multiple possible sugar donors and small-molecule acceptors [14,15,16,31,32]. Moreover, the reactions catalyzed by these enzymes are reversible under physiological conditions, meaning that the same enzyme can also act to cleave the sugar from the small molecule [31].…”

“…GT-1 GTases involved in the synthesis of plant secondary metabolites (sometimes termed UDP glycosyltransferases or UGTs) represent an extreme limit of variable-chemistry promiscuity. A single UGT typically has multiple possible sugar donors and small-molecule acceptors [ 14 , 15 , 16 , 31 , 32 ]. Moreover, the reactions catalyzed by these enzymes are reversible under physiological conditions, meaning that the same enzyme can also act to cleave the sugar from the small molecule [ 31 ].…”