Importance of exposed aromatic residues in chitinase B from Serratia marcescens 2170 for crystalline chitin hydrolysis.
Chitinase B (ChiB) of S. marcescens has five exposed aromatic residues linearly aligned toward the catalytic cleft, Tyr481 and Trp479 in the C-terminal domain, and Trp252, Tyr240 and Phe190 in the catalytic domain. To determine the contribution of these residues to the hydrolysis of crystalline beta-chitin, site-directed mutagenesis, to replace them by alanine, was carried out. The Y481A, W479A, W252A, and Y240A mutations all decreased the binding activity and hydrolyzing activity toward beta-chitin microfibrils. Substitution of Trp residues affected the binding activity more severely than that of Tyr residues. The F190A mutation decreased neither the binding activity nor the hydrolyzing activity. None of the mutations decreased the hydrolyzing activity toward soluble substrates. These results suggest that ChiB hydrolyzes crystalline beta-chitin via a mechanism in which four exposed aromatic residues play important roles, similar to the mechanism of hydrolysis by ChiA of this bacterium, although the directions of hydrolysis of the two chitinases are opposite.
Katouno
Fuminori
F
Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, 8050 Ikarashi-2, Niigata 950-2181.
Taguchi
Masashi
M
Sakurai
Kengo
K
Uchiyama
Taku
T
Nikaidou
Naoki
N
Nonaka
Takamasa
T
Sugiyama
Junji
J
Watanabe
Takeshi
T
D002384:Catalysis; D002686:Chitin / Q000737:chemistry*; D002688:Chitinases / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D020894:Microfibrils / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012706:Serratia marcescens / Q000201:enzymology*; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D017931:DNA Primers; D002686:Chitin; D014443:Tyrosine; D010649:Phenylalanine; D014364:Tryptophan; D002688:Chitinases