PDBsum entry 4lgx

Hydrolase

PDB id: 4lgx

Contents

Protein chain

398 a.a.

Ligands

ACT

GOL ×2

Waters ×593

PDB id:

4lgx

Name:

Hydrolase

Title:

Structure of chitinase d from serratia proteamaculans revealed an unusually constrained substrate binding site

Structure:

Glycoside hydrolase family 18. Chain: a. Engineered: yes

Source:

Serratia proteamaculans. Organism_taxid: 399741. Strain: 568. Gene: spro_2725. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.49Å R-factor: 0.163 R-free: 0.176

Authors:

J.Madhuprakash,A.Singh,S.Kumar,M.Sinha,P.Kaur,S.Sharma,A.R.Podile, T.P.Singh

Key ref:

J.Madhuprakash et al. (2015). Inverse relationship between chitobiase and transglycosylation activities of chitinase-D from Serratia proteamaculans revealed by mutational and biophysical analyses. Sci Rep, 5, 15657. PubMed id: 26493546 DOI: 10.1038/srep15657

Date:

30-Jun-13 Release date: 02-Oct-13

Protein chain

A8GFD6  (A8GFD6_SERP5) -  Glycoside hydrolase family 18 from Serratia proteamaculans (strain 568)

Seq: 426 a.a.

Struc: 398 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.2.1.14 - chitinase.
• Reaction: Hydrolysis of the 1,4-beta-linkages of N-acetyl-D-glucosamine polymers of chitin.

DOI no: 10.1038/srep15657

Sci Rep 5:15657 (2015)

PubMed id: 26493546

Inverse relationship between chitobiase and transglycosylation activities of chitinase-D from Serratia proteamaculans revealed by mutational and biophysical analyses.

J.Madhuprakash, K.B.Bobbili, B.M.Moerschbacher, T.P.Singh, M.J.Swamy, A.R.Podile.

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.