Abstract
Serratia proteamaculans chitinase-D (SpChiD) has a unique combination of hydrolytic and transglycosylation (TG) activities. The TG activity of SpChiD can be used for large-scale production of chito-oligosaccharides (CHOS). The multiple activities (hydrolytic and/or chitobiase activities and TG) of SpChiD appear to be strongly influenced by the substrate-binding cleft. Here, we report the unique property of SpChiD substrate-binding cleft, wherein, the residues Tyr28, Val35 and Thr36 control chitobiase activity and the residues Trp160 and Trp290 are crucial for TG activity. Mutants with reduced (V35G and T36G/F) or no (SpChiDΔ30–42 and Y28A) chitobiase activity produced higher amounts of the quantifiable even-chain TG product with degree of polymerization (DP)-6, indicating that the chitobiase and TG activities are inversely related. In addition to its unprecedented catalytic properties, unlike other chitinases, the single modular SpChiD showed dual unfolding transitions. Ligand-induced thermal stability studies with the catalytically inactive mutant of SpChiD (E153A) showed that the transition temperature increased upon binding of CHOS with DP2–6. Isothermal titration calorimetry experiments revealed the exceptionally high binding affinities for E153A to CHOS with DP2–6. These observations strongly support that the architecture of SpChiD substrate-binding cleft adopted to control chitobiase and TG activities, in addition to usual chitinase-mediated hydrolysis.
Highlights
Require CHOS with a degree of polymerization (DP) ≥ 4, and several enzymatic methods are available to generate CHOS with a DP ≥ 6 using specific chitinases[7,8]
The SpChiD crystal structure (PDB: 4LGX) combined with differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC) data prompted us to test additional mutations to determine the role of the loop (Asn30–Asp42), and the Trp residues in the binding pocket, in TG activity
SpChiD displayed an exceptionally high TG activity, in addition to its other inherent functions, and this can be exploited to increase the yield of long-chain CHOS for biological applications
Summary
Require CHOS with a degree of polymerization (DP) ≥ 4 , and several enzymatic methods are available to generate CHOS with a DP ≥ 6 using specific chitinases[7,8]. There is a need to curtail the hydrolytic activity of SpChiD to improve the yield of TG products, which requires a careful understanding of the role of key residues at the substrate-binding cleft. The SpChiD substrate-binding cleft contains four tryptophan residues (Trp[114], Trp[160], Trp[290] and Trp395) with Trp[160] and Trp[290] flanking the entrance and Trp[114] and Trp[395] situated in the catalytic groove of the binding pocket (Fig. 1A). The spatial alignment of these residues within the proximity of the substrate-binding cleft (PDB: 4LGX) may facilitate ligand binding and control the interplay between hydrolysis and TG activities. The SpChiD crystal structure (PDB: 4LGX) combined with DSC and ITC data prompted us to test additional mutations to determine the role of the loop (Asn30–Asp42), and the Trp residues in the binding pocket, in TG activity. The novel loop and tryptophan residues were highly conserved in 18 of these enzymes, confirming their divergence from other groups (Fig. 1B,C)
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