Plant-derived UDP-glycosyltransferase and Its Molecular Modification

doi:10.13523/j.cb.2105001

China Biotechnology

2021, Vol. 41

Issue (9): 78-91 DOI: 10.13523/j.cb.2105001

Plant-derived UDP-glycosyltransferase and Its Molecular Modification

GUO Fang¹,ZHANG Liang¹,FENG Xu-dong^1,^**(

),LI Chun^1,^2,³

1 Key Laboratory of Medical Molecule Science and Pharmaceutics Engineering, Ministry of Industry and Information Technology, Institute of Biochemical Engineering, Department of Chemical Engineering, School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing 102488, China
2 Key Lab for Industrial Biocatalysis, Ministry of Education, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China
3 Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, China

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Abstract

Glycosylation can increase the structural diversity of plant natural products, and effectively improve their water solubility, pharmacological activity, and bioavailability, which are critical for the drug development of plant natural products. UDP-glycosyltransferases (UGTs) catalyze the transfer of sugar groups from the activated UDP sugar donors to the acceptors to form glycosidic bonds. The glycosylation of natural products is mainly achieved by UGTs in plants. The rapid growth of plant genome and transcriptome data provides an unprecedented opportunity to explore new UGTs. Three methods have been developed to characterize the catalytic function of UGTs: mutant isolation and cloning, direct gene cloning and characterization, and heterologous probe screening of cDNA libraries. As of December 2020, 412 UGTs have been functionally identified. UGTs belong to the GT-1 family and share a unified conserved sequence (plant secondary product glycosyltransferase, PSPG). The structure of UGTs is mainly solved by the X-ray diffraction technology. The GT-B topology of UGTs contains one N-terminal domain and C-terminal domain both with Rossmann (β/α/β)-like folds. The middle cavity becomes the binding regions of the sugar donors and receptors. MODELLER, I-TASSER and SWISS-MODEL are three commonly used software for building three-dimensional structural models of proteins, which have been widely employed in modeling UGTs structure. UGTs exhibit an inverting catalytic mechanism, usually associated with a bimolecular nucleophilic substitution reaction (S_N2), and the highly conserved catalytic dimer (His-Asp) in the active site is essential for the glycosylation activity of UGTs. However, most plant UGTs show low catalytic activity, stability, and substrate specificity, which has limited their industrial application. Recently, the improvement of the catalytic properties of UGTs by molecular modification has achieved significant progress. This review summarizes the following five modification methods for UGTs. The first method, domain replacement combined with site-directed mutation, is mostly used between UGTs with high sequence similarity or between enzymes of the same family to produce new UGTs with different functions. Because they have a highly conservative three-dimensional structure, including the N-terminal domain that recognizes the acceptors and the C-terminal domain that recognizes the UDP-sugar donors, the specificity of the sugar-donor and acceptor substrates is quite different. The key amino acids in or near the catalytic activity pocket usually determine or directly affect the catalytic activity and substrate specificity of UGTs. After multiple sequence alignments, the strategy of replacing non-consensus amino acid residues with consensus sequences at each position is the second method, called activity-based sequence conservation analysis (ASCA), which can improve the substrate specificity and catalytic activity of UGTs. Through three-dimensional structure simulation and protein-ligand interaction analysis, the structural characteristics of active sites and their effects on functions are deeply studied. Rational design based on the structure-function relationship is the third method and has become a powerful means of molecular modification for UGTs. Directed evolution does not require an in-depth understanding of the spatial structure and catalytic mechanism of UGTs. The fourth method is to simulate the natural biological evolution process by utilizing error-prone PCR or semi-rational saturation mutation techniques, and screening mutations with optimized performance. The last one, the structure-guided directed evolution method, such as iterative saturation mutagenesis (ISM) and combined active site saturation test (CAST), bears the advantages of both rational design and directed evolution. Overall, this review delineates the mining strategies, properties, three-dimensional structure, and catalytic mechanism of plant-derived UGTs, and summarizes the strategies of molecular modification for UGTs, including rational design and directed evolution. It provides guidance for the industrialization of plant natural product glycosides by enzymatic synthesis.

Key words： UDP-glycosyltransferase Natural products Site-directed mutation Directed evolution High-throughput screening

Received: 02 May 2021 Published: 30 September 2021

ZTFLH:

Q816Q814.9

Corresponding Authors: Xu-dong FENG E-mail: xd.feng@bit.edu.cn

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	Fang GUO
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	Chun LI

Cite this article:

GUO Fang,ZHANG Liang,FENG Xu-dong,LI Chun. Plant-derived UDP-glycosyltransferase and Its Molecular Modification. China Biotechnology, 2021, 41(9): 78-91.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2105001 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I9/78

Table 1 Plant-derived UGTs with structural analysis

Fig.1 Three-dimensional structure of UGTs with GT-B folding

Fig.2 The reaction mechanism of UGTs (a)S_N2 type reaction (b)S_Ni type reaction B: Catalytic residues in UGTs; X: O, N, S, C atoms that can generate glycosidic bonds

Fig.3 The rational design of UDP-glycosyltransferases (a) Establishment of three-dimensional simulation structure and molecular docking (b) Force analysis to determine key amino acid residues (c) Multiple sequence alignment

Table 2 Summary of high-throughput screening methods for glycosyltransferases


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