Organization of glycoside hydrolase (GH) families into clans expands the utility of information on catalytic mechanisms of member enzymes. This issue was examined for GH27 and GH36 through biochemical analysis of GH36 alpha-galactosidase from Thermotoga maritima (TmGalA). Catalytic residues in TmGalA were inferred through structural homology with GH27 members to facilitate design of site-directed mutants. Product analysis confirmed that the wild type (WT) acted with retention of anomeric stereochemistry, analogous to GH27 enzymes. Conserved acidic residues were confirmed through kinetic analysis of D327G and D387G mutant enzymes, azide rescue, and determination of azide rescue products. Mutation of Asp327 to Gly resulted in a mutant that had a 200-800-fold lower catalytic rate on aryl galactosides relative to the WT enzyme. Azide rescue experiments using the D327G enzyme showed a 30-fold higher catalytic rate compared to without azide. Addition of azide to the reaction resulted in formation of azide beta-d-galactopyranoside, confirming Asp327 as the nucleophilic residue. The Asp387Gly mutation was 1500-fold catalytically slower than the WT enzyme on p-nitrophenyl alpha-d-galactopyranoside. Analysis at different pH values produced a bell-shaped curve of the WT enzyme, but D387G exhibited higher activity with increasing pH. Catalyzed reactions with the D387G mutant in the presence of azide resulted in formation of azide alpha-d-galactopryanoside as the product of a retaining mechanism. These results confirm that Asp387 is the acid/base residue of TmGalA. Furthermore, they show that the biochemical characteristics of GH36 TmGalA are closely related to GH27 enzymes, confirming the mechanistic commonality of clan GH-D members.

While screening of small-molecular metabolites produced by most cultivatable microorganisms often results in rediscovery of known compounds, genome-mining programs allow to harness much greater chemical diversity and result in discovery of new molecular scaffolds. Here we report genome-guided identification of a new antibiotic klebsazolicin (KLB) from Klebsiella pneumoniae that inhibits growth of sensitive cells by targeting ribosome. A member of ribosomally-synthesized post-translationally modified peptides (RiPPs), KLB is characterized by the presence of unique N-terminal amidine ring essential for its activity. Biochemical in vitro studies indicate that KLB inhibits ribosome by interfering with translation elongation. Structural analysis of the ribosome-KLB complex reveals the compound bound in the peptide exit tunnel overlapping with the binding sites of macrolides or streptogramins-B. KLB adopts compact conformation and largely obstructs the tunnel. Engineered KLB fragments retain in vitro activity and can serve as a starting point for the development of new bioactive compounds.