Crystal structures of Vibrio harveyi chitinase A complexed with chitooligosaccharides: implications for the catalytic mechanism.
This research describes four X-ray structures of Vibrio harveyi chitinase A and its catalytically inactive mutant (E315M) in the presence and absence of substrates. The overall structure of chitinase A is that of a typical family-18 glycosyl hydrolase comprising three distinct domains: (i) the amino-terminal chitin-binding domain; (ii) the main catalytic (alpha/beta)(8) TIM-barrel domain; and (iii) the small (alpha+beta) insertion domain. The catalytic cleft of chitinase A has a long, deep groove, which contains six chitooligosaccharide ring-binding subsites (-4)(-3)(-2)(-1)(+1)(+2). The binding cleft of the ligand-free E315M is partially blocked by the C-terminal (His)(6)-tag. Structures of E315M-chitooligosaccharide complexes display a linear conformation of pentaNAG, but a bent conformation of hexaNAG. Analysis of the final 2F(o)-F(c) omit map of E315M-NAG6 reveals the existence of the linear conformation of the hexaNAG at a lower occupancy with respect to the bent conformation. These crystallographic data provide evidence that the interacting sugars undergo conformational changes prior to hydrolysis by the wild-type enzyme.
Songsiriritthigul
Chomphunuch
C
School of Biochemistry, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
Pantoom
Supansa
S
Aguda
Adeleke H
AH
Robinson
Robert C
RC
Suginta
Wipa
W
D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D002686:Chitin / Q000737:chemistry; D002688:Chitinases / Q000737:chemistry*; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray / Q000379:methods; D006868:Hydrolysis; D008968:Molecular Conformation; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014733:Vibrio / Q000201:enzymology
D009844:Oligosaccharides; D002686:Chitin; D002688:Chitinases