TY - JOUR
T1 - Crystal structures of Vibrio harveyi chitinase A complexed with chitooligosaccharides
T2 - Implications for the catalytic mechanism
AU - Songsiriritthigul, Chomphunuch
AU - Pantoom, Supansa
AU - Aguda, Adeleke H.
AU - Robinson, Robert C.
AU - Suginta, Wipa
N1 - Funding Information:
This work is financially supported by The Thailand Research Fund (TRF) and The Thai Commission on Higher Education through Research Career Development Grant (RMU4980028), and the National Synchrotron Research Centre (NSRC), Thailand. R.C.R. and A.H.A. thank the Institute of Molecular and Cell Biology, Biopolis, Singapore and A ∗ STAR, Singapore for their support.
PY - 2008/6
Y1 - 2008/6
N2 - 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 (α/β)8 TIM-barrel domain; and (iii) the small (α + β) 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 2Fo - Fc 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.
AB - 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 (α/β)8 TIM-barrel domain; and (iii) the small (α + β) 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 2Fo - Fc 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.
KW - Chitin oligosaccharide
KW - Chitinase
KW - Crystal structure
KW - The slide-and-bend mechanism
KW - Vibrio harveyi
UR - http://www.scopus.com/inward/record.url?scp=44649153432&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=44649153432&partnerID=8YFLogxK
U2 - 10.1016/j.jsb.2008.03.008
DO - 10.1016/j.jsb.2008.03.008
M3 - Article
C2 - 18467126
AN - SCOPUS:44649153432
VL - 162
SP - 491
EP - 499
JO - Journal of Structural Biology
JF - Journal of Structural Biology
SN - 1047-8477
IS - 3
ER -