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

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