Structural dissection of unnatural ginsenoside-biosynthetic UDP-glycosyltransferase Bs-YjiC from Bacillus subtilis for substrate promiscuity

https://doi.org/10.1016/j.bbrc.2020.11.104Get rights and content

Highlights

  • Crystal structure of a promiscuous glycosyltransferase Bs-YjiC was solved.

  • Binary structure of Bs-YjiC and uridine diphosphate was solved.

  • Molecular docking was performed to obtain sugar acceptor-bound Bs-YjiC complexes.

Abstract

Glycosylation catalyzed by uridine diphosphate-dependent glycosyltransferases (UGT) contributes to the chemical and functional diversity of a number of natural products. Bacillus subtilis Bs-YjiC is a robust and versatile UGT that holds potentials in the biosynthesis of unnatural bioactive ginsenosides. To understand the molecular mechanism underlying the substrate promiscuity of Bs-YjiC, we solved crystal structures of Bs-YjiC and its binary complex with uridine diphosphate (UDP) at resolution of 2.18 Å and 2.44 Å, respectively. Bs-YjiC adopts the classical GT-B fold containing the N-terminal and C-terminal domains that accommodate the sugar acceptor and UDP-glucose, respectively. Molecular docking indicates that the spacious sugar-acceptor binding pocket of Bs-YjiC might be responsible for its broad substrate spectrum and unique glycosylation patterns toward protopanaxadiol–(PPD) and PPD-type ginsenosides. Our study reveals the structural basis for the aglycone promiscuity of Bs-YjiC and will facilitate the protein engineering of Bs-YjiC to synthesize novel bioactive glycosylated compounds.

Introduction

Glycosylation mediated by uridine diphosphate-dependent glycosyltransferase (UGT) catalyzes the transfer of sugar moieties from activated nucleotide diphosphate (NDP) sugars to sugar acceptors. This process is one of the most common and important modifications in natural compound biosynthesis [1]. Glycosylation is the final biosynthetic step of many natural products which can enhance their solubility, stability, and bioactivity [2]. UGTs are one of the largest supergene families that widely distributed in animals, plants, and microbes [3]. To date, over 25,000 UGTs have been deposited to the carbohydrate-active enzymes database (CAZy, http://www.cazy.org/). UGTs adopt the canonical GT-B fold with two similar Rossmann-like domains that are linked by a loop [4]. The N-terminal domain is responsible for the binding of the sugar acceptor and the C-terminal domain provides the NDP-sugar-binding site.

Ginsenosides are the principal bioactive compounds isolated from Panax ginseng (a perennial herb crowned the ‘King of all Herbs’ in Eastern Asia) [5]. These highly valuable phytochemicals are a group of dammarane-type tetracyclic triterpene saponins. Ginsenosides exhibit diverse biological effects such as anticancer, antitumor, antiaging, and immunostimulatory activities [6]. More than 140 ginsenosides have been isolated from P. ginseng. These natural products are mainly categorized into protopanaxadiol–(PPD) and protopanaxatriol–(PPT) types according to their triterpene skeleton [7]. Glycosylation catalyzed by UGTs is the last step of ginsenosides biosynthesis, which also determines their structural and functional diversity [8]. For naturally-occurring PPD-type ginsenosides, the sugar moieties are only linked to the C3- and/or C20–OH of PPD because plant UGTs can only catalyze glycosylation of PPD on these positions. A microbial UGT from Bacillus subtilis named Bs-YjiC is a robust and promiscuous UGT that can catalyze the glycosylation using a considerable number of structurally diverse natural and unnatural products [[9], [10], [11]]. More interestingly, Bs-YjiC can catalyze the consecutive glycosylation on C3- then C12–OH of PPD to produce ginsenoside Rh2 (2.1%) and then unnatural ginsenoside F12 (97.8%) (Fig. 1A) [12,13]. Therefore, the identification of Bs-YjiC greatly expands the structural and functional diversity of ginsenosides, which should benefit the development of new compound leads.

Structural information of Bs-YjiC is demanded in order to understand the mechanism underlying the substrate promiscuity and regioselectivity. Up to date, forty-four UGT crystal structures are available in the CAZy database. Among these enzymes, only UGT51 from Saccharomyces cerevisiae and UGT74AC1 from Siraitia grosvenorii could selectively glycosylate PPD on C3–OH to synthesize ginsenoside Rh2 [14,15]. Nevertheless, neither of them can catalyze C12–OH glycosylation. Moreover, Bs-YjiC shares very low amino acid sequence identify to UGT51 and UGT74AC1 (<20%), making it very difficult to predict the Bs-YjiC structure via modeling approaches. In the present study, the crystal structure of Bs-YjiC and its complex with UDP were solved. Moreover, the molecular mechanism for the interactions between Bs-YjiC and PPD and ginsenoside Rh2 was determined by molecular docking simulations.

Section snippets

Expression and purification of Bs-YjiC

The open reading frame of Bs-YjiC (GenBank accession no: NP_389104.1) was subcloned into the pET28a vector and fused with an N-terminal His6 tag and a tobacco etch virus (TEV) protease cleavage site. Expression and purification of the His6–tag Bs-YjiC were performed as described previously [10]. Briefly, recombinant Escherichia coli BL21 (DE3) strains harboring pET28a-Bs-YjiC were induced with 0.3 mM isopropyl β-d-thiogalactopyranoside. After incubated at 16 °C for 16 h, the recombinant cells

Overall structure of Bs-YjiC

The 46-kDa Bs-YjiC was purified to apparent homogeneity, which was found to exist as a monomer in solution as revealed by SDS–PAGE analysis and static light scattering (Figure S1). Bs-YjiC was also successfully crystallized in space group I222 which contains one molecule in an asymmetric unit. The crystal structure of Bs-YjiC (residues 6–387) as well as its binary complex with UDP was solved and refined at 2.18- and 2.44 Å resolution, respectively. Detailed data collection and refinement

Declaration of competing interest

The authors declare no conflict of interest and this work and related studies are not in press or submitted to other journals.

Acknowledgements

This work was supported by the National Natural Science Foundation of China Grant (no. 21702226), Natural Science Foundation of Hubei Province (No. 2019CFB386), and the China Postdoctoral Science Foundation (no. 2019M662575 and 2020M682381). The synchrotron data collection was conducted at beam line TPS-5A and BL15A1 of National Synchrotron Radiation Research Center (NSRRC, Taiwan).

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