Structural And Biochemical Insights Into The Substrate Binding And Catalytic Mechanism Of Chitosanase OU01

Chitosan is a polycationic saccharide and recent studies have demonstrated that the chitooligomer derived from chitosan has a variety of bioactivities including antimicrobial, antioxidant inhibition of tumor growth. Previous antitumor activity study for chitooligomers as a function of molecular weight revealed that chitooligomers with a molecular weight range 1.5-5.5 kDa demonstrated effectively inhibition of tumor cell growth. Therefore, it is urgent and necessary to establish a strategy to produce well-defined chitooligomer through the hydrolysis of chitosan. Hydrolysis of chitosan can be achieved through either chemical or enzymatic approaches. Chemical-based hydrolysis of chitosan has several disadvantages over enzymatic-based approaches. For example, extensive use of acid in chemical based approaches resulted in the production of significant amounts of acidic waste, and associated production of other toxic wastes. In contrast, the enzymatic-based hydrolysis of chitosan is regarded as an environmentally friendly approach. Obviously, a suitable chitosanase is the determinant for enzymatic-based approach. Although several structures for chitosanase have been determined, detailed knowledge for the overall hydrolysis and catalytic mechanism of chitosanase is still lack, which is critical for the design of novel enzymes for well-defined chito- oligosaccharides generation. To address this question, a high-resolution structure of the chitosanase OU01-(GlcN)6 complex was determined and revelant research on substrate-binding and catalytic mechanism was carried out.1. The gene of chitosanase OU01 was cloned from chromosomal DNA of Microbacterium sp. and inserted into the pGEX-6p-1 vector. The resulting expression plasmid was transformed into E. coli BL21 (DE3). Chitosanase OU01 was expressed successfully with the IPTG induction. The 30 kDa protein with high purity was obtained using a three-step purificant strategy (glutathione-sepharose affinity, precission cleavage followed by gel filtration chromatography).2. The specific activity for chitosanase OU01 was 476.6 U/mgprotein, and its Km and Kcat values were determined as 4.197 mg/ml,12,297 min-1, respectively. The dominant end product of chitosan (DDA>90%) hydrolyzed by chitosanase OU01 were chitodimer and chitotrimer. And the time course of the degradation of polymeric chitosan (DDA>99%) by chitosanase OU01 showed that the degradation reaction followed an initially rapid phase and a following slower second phase.3. Through amino acid sequence alignment, two key residues:Glu25 and Asp43 in chitosanase OU01 were determined. And inactive mutation E25A and D43A were generated by site-directed mutagenesis. The co-crystal of the mutant -hexameric glucosamine complex was diffracted by X-ray. The structures of the final complex have been deposited in the PDB with accession number 4OLT and 4QWP.4. The complex structure provided the structural evidence for chitosanase OU01 follows an "inverting" catalytic mechanism. And the role of two key residues was clarified.5. Interactions formed between the cleft and hexameric glucosamine were analyzed and several key residues in cleft involved in substrate binding were determined.6. Structural, biophysical analysis and molecular modeling strongly indicated that chitosanase OU01 has specificity for GlcN in the -2 position and can accommodate GlcNAc at other sites. The chitosanase OU01 should be classified as the D-X-X ((-2)-(-1)-(+1)) type chitosanase.7. A substrate binding site (Asp40) out of the binding cleft was determined by surface electrostatic analysis and mutant activity comparison.8. Based on the mutagenesis assay and structural analysis, critical residues could be divided into three categories. In addition, a three-step overall binding mechanism was proposed.9. Two significant structural features are summarized for non-processive chitosanase:1). open-closed-open protein conformation change by substrate binding; 2). polar residues constitute the substrate binding cleft.Taken together, this study provides novel structural insights into the understanding of overall hydrolysis mechanism for non-processive chitsoanase, and also will facilitate the design of new enzymes used for industrial purpose.