Structure of the D142N mutant of the family 18 chitinase ChiB from Serratia marcescens and its complex with allosamidin.
Catalysis by ChiB, a family 18 chitinase from Serratia marcescens, involves a conformational change of Asp142 which is part of a characteristic D(140)XD(142)XE(144) sequence motif. In the free enzyme Asp142 points towards Asp140, whereas it rotates towards the catalytic acid, Glu144, upon ligand binding. Mutation of Asp142 to Asn reduced k(cat) and affinity for allosamidin, a competitive inhibitor. The X-ray structure of the D142N mutant showed that Asn142 points towards Glu144 in the absence of a ligand. The active site also showed other structural adjustments (Tyr10, Ser93) that had previously been observed in the wild-type enzyme upon substrate binding. The X-ray structure of a complex of D142N with allosamidin, a pseudotrisaccharide competitive inhibitor, was essentially identical to that of the wild-type enzyme in complex with the same compound. Thus, the reduced allosamidin affinity in the mutant is not caused by structural changes but solely by the loss of electrostatic interactions with Asp142. The importance of electrostatics was further confirmed by the pH dependence of catalysis and allosamidin inhibition. The pH-dependent apparent affinities for allosamidin were not correlated with k(cat), indicating that it is probably better to view the inhibitor as a mimic of the oxazolinium ion reaction intermediate than as a transition state analogue.
Vaaje-Kolstad
Gustav
G
Department of Chemistry and Biotechnology, Agricultural University of Norway, PO Box 5040, N-1432 Aas, Norway.
Houston
Douglas R
DR
Rao
Francesco V
FV
Peter
Martin G
MG
Synstad
Bjørnar
B
van Aalten
Daan M F
DM
Eijsink
Vincent G H
VG
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000737:chemistry*; D001667:Binding, Competitive; D002384:Catalysis; D002688:Chitinases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012706:Serratia marcescens / Q000201:enzymology* / Q000235:genetics; D014312:Trisaccharides / Q000737:chemistry*
D004791:Enzyme Inhibitors; D014312:Trisaccharides; C054851:allosamidin; C482346:ChiB protein, Serratia marcescens; D002688:Chitinases; D000117:Acetylglucosamine