Inverse relationship between chitobiase and transglycosylation activities of chitinase-D from Serratia proteamaculans revealed by mutational and biophysical analyses.
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.
Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Gachibowli, Hyderabad, India.
Jogi
J
Madhuprakash
School of Chemistry, University of Hyderabad, Gachibowli, Hyderabad, India.
Kishore Babu
KB
Bobbili
Institute for Biology and Biotechnology of Plants, WWU, Münster University, Münster, Germany.
Bruno M
BM
Moerschbacher
Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India.
Tej Pal
TP
Singh
School of Chemistry, University of Hyderabad, Gachibowli, Hyderabad, India.
Musti J
MJ
Swamy
Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Gachibowli, Hyderabad, India.
Appa Rao
AR
Podile
D000118:Acetylglucosaminidase / Q000378:metabolism*; D000595:Amino Acid Sequence; D002688:Chitinases / Q000737:chemistry / Q000378:metabolism*; D004795:Enzyme Stability; D006031:Glycosylation; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D012705:Serratia / Q000201:enzymology*
D002688:Chitinases; D000118:Acetylglucosaminidase