Modeled 3D-Structures of Proteobacterial Transglycosylases from Glycoside Hydrolase Family 17 Give Insight in Ligand Interactions Explaining Differences in Transglycosylation Products

Javier A Linares-Pastén,Gudmundur O Hreggvidsson,Hildegard Watzlawick,Olafur H Fridjonsson,Lilja Björk Jonsdottir,Eva Nordberg Karlsson

doi:10.3390/app11094048

Javier A Linares-Pastén, Gudmundur O Hreggvidsson + Show 4 more

Open Access

https://doi.org/10.3390/app11094048

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Journal: Applied sciences	Publication Date: Apr 29, 2021
Citations: 3	License type: CC BY 4.0

Affiliation: Lund University, University of Stuttgart

Abstract

The structures of glycoside hydrolase family 17 (GH17) catalytic modules from modular proteins in the ndvB loci in Pseudomonas aeruginosa (Glt1), P. putida (Glt3) and Bradyrhizobium diazoefficiens (previously B. japonicum) (Glt20) were modeled to shed light on reported differences between these homologous transglycosylases concerning substrate size, preferred cleavage site (from reducing end (Glt20: DP2 product) or non-reducing end (Glt1, Glt3: DP4 products)), branching (Glt20) and linkage formed (1,3-linkage in Glt1, Glt3 and 1,6-linkage in Glt20). Hybrid models were built and stability of the resulting TIM-barrel structures was supported by molecular dynamics simulations. Catalytic amino acids were identified by superimposition of GH17 structures, and function was verified by mutagenesis using Glt20 as template (i.e., E120 and E209). Ligand docking revealed six putative subsites (−4, −3, −2, −1, +1 and +2), and the conserved interacting residues suggest substrate binding in the same orientation in all three transglycosylases, despite release of the donor oligosaccharide product from either the reducing (Glt20) or non-reducing end (Glt1, Gl3). Subsites +1 and +2 are most conserved and the difference in release is likely due to changes in loop structures, leading to loss of hydrogen bonds in Glt20. Substrate docking in Glt20 indicate that presence of covalently bound donor in glycone subsites −4 to −1 creates space to accommodate acceptor oligosaccharide in alternative subsites in the catalytic cleft, promoting a branching point and formation of a 1,6-linkage. The minimum donor size of DP5, can be explained assuming preferred binding of DP4 substrates in subsite −4 to −1, preventing catalysis.

Highlights

Leloir glycosyl transferases (GTs) that utilize nucleotide-activated monosaccharides as donor substrates are the most common enzymes to catalyze glycosylation reactions in nature [1]
CGTases can be used for the synthesis of alkyl glycosides [7,8]
This, together with molecular dynamics refinement, resulted in acceptable models according to several protein-structure validation approaches, including Ramachandran analysis (Table 1) and Z-scores

Summary

Introduction

Leloir glycosyl transferases (GTs) that utilize nucleotide-activated monosaccharides as donor substrates are the most common enzymes to catalyze glycosylation reactions in nature [1]. GTs are, not well suited for in vitro glycosylation reactions, due to the requirement of expensive nucleotide-activated glycosyl donors, as well as reported recombinant expression difficulties. Instead, retaining glycoside hydrolases (GH), with high transglycosylation activity (often referred to as transglycosylases) are ideal candidates for glycosylation reactions, due to natural abundance, robustness and wide acceptor specificity [2,3]. Oligo- or polysaccharide acceptors react instead of water, resulting in a new glycosidic bond. Some examples with main specificities for α-glucosyl groups, include cyclomaltodextrin glucanotransferases (CGTases in family GH13) [5,6]. CGTases can be used for the synthesis of alkyl glycosides [7,8]

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