Crystal Structure And Inhibitor Screening Of Chitin Deacetylase From Puccinia Striiformis F. Sp. Tritici

Puccinia striiformis f.sp.tritici（Pst）is one of the most serious plant pathogenic fungi,which is the main cause of wheat stripe rust.At present,there is no efficient fungicides to control Pst.The chitin deacetylase Pst₁3661 has been reported to be a key pathogenic factor for its escape from plant immunity.The loss of its activity hampers the growth and development of Pst and greatly reduces the pathogenicity of Pst.As there is no homologue of Pst₁3661 in human and plants,inhibiting the activity of Pst₁3661 by small molecular compounds shall be a human-and plant-safe strategy to control wheat stripe rust.In this regard,this work has achieved the following accomplishment:（1）The Pst₁3661 protein was recombinantly expressed in the yeast strain Pichia pastoris and the recombinant Pst₁3661 was purified by nickel affinity chromatography and gel filtration chromatography.The high purity of Pst₁3661 was obtained.（2）Benzhydroxamic acid（BHA）was discovered to significantly inhibit the deacetylation activity of Pst₁3661 by means of fluorescamine method.The optimum reaction conditions were p H 7.5 and 30℃ for 20 min.The inhibition rates of BHA at a concentration of 20μM and 100μM were（57.7±2.0）% and（100±5.5）%,respectively.And the Ki value of BHA was determined to be 11.96 μM。（3）Crystal structure of Pst₁3661 was obtained by hanging-drop vapordiffusion crystallization experiments and resolved at a resolution of 1.96 ? by molecular replacement using the chitin deacetylase from Colletotrichum lindemuthianum,Cl CDA,as a template.The structural architecture of Pst₁3661 shows typical characteristics of CE-4 family enzymes.It has a（β/α）7 barrel as a core for catalysis and a zinc ion in the center of the active pocket,which forms a stable HHD triplet.（4）The crystal structure of Pst₁3661 complexed with the inhibitor BHA was obtained and resolved at a resolution of 1.61 ?.The complex structure shows that hydroxyl and carbonyl groups of BHA hydroxamic acid moiety form hydrogen bonds with the zinc ion that is coordinated by Asp34,His183 and Tyr128.The hydrophobicity of the benzene ring is well suited in the hydrophobic pocket that is formed by hydrophobic tryptophan and leucine residues.（5）Nine BHA derivatives were evaluated by their inhibitory activity of Pst₁3661.Two of them exhibited BHA-comparable inhibition activity with Ki values of 27.58 μM and 10.75μM,respectively.The crystal structures of these compounds bound to Pst₁3661 indicate that the hydroxamic acid moiety is the major contributor to interact with the active center,in a way similar to BHA.In conclusion,this work has revealed for the first time the pathogenic effector Pst₁3661with regard to its biochemical properties,crystal structures and inhibitory mechanism,providing the core information for developing Pst₁3661-targeting agents in the control of wheat stripe rust,which is the most serious threat to wheat production.