Molecular Basis of Pseudomonas syringae pv actinidiae Levansucrase Inhibition by a Multivalent Iminosugar

Levansucrases are a class of polysaccharide-processing enzymes widely distributed among plant pathogenic bacteria, such as Pseudomonas syringae and Erwinia amylovora. Therefore, the modulation of levansucrase activity could represent a new strategy to reduce the microbial survival of such bacteria. Herein, we identified a tetravalent pyrrolidine iminosugar (TPIS) as the first levansucrase inhibitor described to date. TPIS reversibly inhibits sucrose hydrolysis and levan polymerization of levansucrase derived from different bacterial genotypes of P. syringae, showing competitive behavior and an inhibition constant (Ki) in the micromolar range. Interestingly, the monovalent pyrrolidine iminosugar (PIS) analogue shows negligible inhibition, suggesting that multivalency plays a pivotal role in the interaction with levansucrase. To gain insight into the binding mechanism, the X-ray crystal structures of the beta levansucrase isoform from P. syringae pv actinidiae (Psa) in its native form and in complex with TPIS were solved, confirming TPIS as a competitive inhibitor of levansucrases. Only a portion of TPIS, corresponding to one chain of the tetravalent iminosugar derivative, was visible in the electron density maps. Nevertheless, our structural data provided an adequate comprehension of the inhibitor/enzyme interactions, sufficient to exclude some of the possible inhibition mechanisms justifying a multivalent effect and pave the way for the development of new, more potent inhibitors.