Structural insights into the catalytic mechanism of a family 18 exo-chitinase

被引:394
作者
van Aalten, DMF [1 ]
Komander, D
Synstad, B
Gåseidnes, S
Peter, MG
Eijsink, VGH
机构
[1] Univ Dundee, Dept Biochem, Wellcome Trust Bioctr, Dundee DD1 5EH, Scotland
[2] Agr Univ Norway, Dept Chem & Biotechnol, N-1432 As, Norway
[3] Univ Potsdam, Inst Organ Chem & Struct Anal, D-14476 Golm, Germany
关键词
D O I
10.1073/pnas.151103798
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Chitinase B (Chill) from Serratia marcescens is a family 18 exo-chitinase whose catalytic domain has a TIM-barrel fold with a tunnel-shaped active site. We have solved structures of three Chill complexes that reveal details of substrate binding, substrate-assisted catalysis, and product displacement. The structure of an inactive Chill mutant (E144Q) complexed with a pentameric substrate (binding in subsites -2 to +3) shows closure of the "roof" of the active site tunnel. It also shows that the sugar in the -1 position is distorted to a boat conformation, thus providing structural evidence in support of a previously proposed catalytic mechanism. The structures of the active enzyme complexed to allosamidin (an analogue of a proposed reaction intermediate) and of the active enzyme soaked with pentameric substrate show events after cleavage of the glycosidic bond. The latter structure shows reopening of the roof of the active site tunnel and enzyme-assisted product displacement in the +1 and +2 sites, allowing a water molecule to approach the reaction center. Catalysis is accompanied by correlated structural changes in the core of the TIM barrel that involve conserved polar residues whose functions were hitherto unknown. These changes simultaneously contribute to stabilization of the reaction intermediate and alternation of the pKa of the catalytic acid during the catalytic cycle.
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页码:8979 / 8984
页数:6
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