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

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.

Exploring a novel β-1,3-glucanosyltransglycosylase, MlGH17B, from a marine Muricauda lutaonensis strain for modification of laminari-oligosaccharides

“…The lack of hits is judged to be due to that these enzymes are deposited in their original multi-modular form, resulting in low query coverage. To further display similarities and differences, an alignment of the catalytic modules of the characterized bacterial enzymes, in the sequence-format used for recombinant production ( Linares-Pastén et al. 2021 ), was generated.…”

“…2015 ). Homology models are, however, available for three bacterial transglycosylases: Glt1 from P. aeruginosa , Glt3 from P. putida, and Glt20 from B, diazoefficien s ( Linares-Pastén et al. 2021 ).…”

“…Structural superimposition of Ml GH17B with Rm Bgt17A, Glt1, Glt3, and Glt20 (as well as docking experiments) demonstrated that despite a low sequence similarity, Ml GH17B, Glt20, and Rm Bgt17A, shared an overall similar active site architecture, with the exception that Ml GH17B and Rm Bgt17A has five subsites consisting of three glycone subsites (−3, −2, and −1) interacting with the donor and two aglycone subsites (+1, +2) interacting with the acceptor, while the active site of Glt20 is comprised of six potential subsites, with one additional glycone subsite (−4, −3, −2, −1, +1, +2) ( Linares-Pastén et al. 2021 ).…”

“…The transglycosylases are few, and to date the identified bacterial GH17-transglycosylases all originate from proteobacteria from soil habitats ( Hreggvidsson et al. 2011 ; Linares-Pastén et al. 2021 ).…”

Assessment of IsPETase-Assisted Depolymerization of Terephthalate Aromatic Polyesters and the Effect of the Thioredoxin Fusion Domain

“…The calculations were performed by applying the OPLS-AA/L force field [49], periodic boundaries, 8-Å cut-off of short-range electrostatic and van der Waals forces, and long-range forces calculated by the particle mesh Ewald method. The simulations consisted of three steps: energy minimization, equilibrations, and production, as described in a previous publication [50]. After 500 ns of production, the root-mean-square deviations (RMSDs) of the alpha carbons and root-mean-square fluctuation (RMSF) were calculated.…”