Serratia marcescens chitinases with tunnel-shaped substrate-binding grooves show endo activity and different degrees of processivity during enzymatic hydrolysis of chitosan.
The modes of action of three family 18 chitinases (ChiA, ChiB, and ChiC) from Serratia marcescens during the degradation of a water-soluble polymeric substrate, chitosan, were investigated using a combination of viscosity measurements, reducing end assays, and characterization of the size-distribution of the oligomeric products. All three enzymes yielded a fast reduction in molecular weight of the chitosan substrate at a very early stage of hydrolysis, which is typical for endo-acting enzymes. For ChiA and ChiB, this is inconsistent with the previously proposed exo-attack mode of action. The main difference between ChiA, ChiB, and ChiC is the degree of processivity. ChiC is an endo enzyme with no apparent processivity. ChiA and ChiB are processive enzymes in which the substrate remains bound to the active cleft after successful hydrolysis and is moved along for the next hydrolysis to occur. ChiA and ChiB perform on average 9.1 and 3.4 cleavages, respectively, for the formation of each enzyme-substrate complex. ChiA and ChiB have deep, tunnel-like substrate-binding grooves. The demonstration of endo activity shows that substrate binding must involve the temporary restructuring of the loops that make up the roofs of the substrate-binding grooves, similar to what has been proposed for cellobiohydrolase Cel6A. The data suggest that the exo-type of activity observed for ChiA and ChiB during the degradation of solid crystalline chitin is due to the better accessibility of chain ends, rather than intrinsic enzyme properties.
Sikorski
Pawel
P
Department of Physics, Norwegian University of Science and Technology, 7491 Trondheim, Norway. pawel.sikorski@phys.ntnu.no
Sørbotten
Audun
A
Horn
Svein J
SJ
Eijsink
Vincent G H
VG
Vårum
Kjell M
KM
D001426:Bacterial Proteins / Q000737:chemistry*; D002688:Chitinases / Q000737:chemistry*; D048271:Chitosan / Q000737:chemistry; D006868:Hydrolysis; D008956:Models, Chemical; D008970:Molecular Weight; D012706:Serratia marcescens / Q000201:enzymology*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D048271:Chitosan; D002688:Chitinases