4LGX

Structure of Chitinase D from Serratia proteamaculans revealed an unusually constrained substrate binding site

Summary for 4LGX

• Entry DOI: 10.2210/pdb4lgx/pdb
• Related: 3QOK
• Descriptor: Glycoside hydrolase family 18, ACETATE ION, GLYCEROL, ... (4 entities in total)
• Functional Keywords: tim barrel, hydrolase
• Biological source: Serratia proteamaculans
• Total number of polymer chains: 1
• Total formula weight: 45016.61

Authors

Madhuprakash, J.,Singh, A.,Kumar, S.,Sinha, M.,Kaur, P.,Sharma, S.,Podile, A.R.,Singh, T.P. (deposition date: 2013-06-30, release date: 2013-10-02, Last modification date: 2024-11-06)

Primary citation

Madhuprakash, J.,Bobbili, K.B.,Moerschbacher, B.M.,Singh, T.P.,Swamy, M.J.,Podile, A.R.
Inverse relationship between chitobiase and transglycosylation activities of chitinase-D from Serratia proteamaculans revealed by mutational and biophysical analyses.
Sci Rep, 5:15657-15657, 2015

PubMed 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.

PubMed: 26493546
DOI: 10.1038/srep15657

Experimental method

X-RAY DIFFRACTION (1.49 Å)