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Aromatic Residues in the Catalytic Center of Chitinase A from Serratia marcescens Affect Processivity, Enzyme Activity, and Biomass Converting Efficiency*

By Henrik Zakariassen, Berit Bjugan Aam, Svein J. Horn, Kjell M. Vårum, Morten Sørlie and Vincent G. H. Eijsink

Abstract

The processive Serratia marcescens chitinases A (ChiA) and B (ChiB) are thought to degrade chitin in the opposite directions. A recent study of ChiB suggested that processivity is governed by aromatic residues in the +1 and +2 (aglycon) subsites close to the catalytic center. To further investigate the roles of aromatic residues in processivity and to gain insight into the structural basis of directionality, we have mutated Trp167, Trp275, and Phe396 in the -3, +1, and +2 subsites of ChiA, respectively, and characterized the hydrolytic activities of the mutants toward β-chitin and the soluble chitin-derivative chitosan. Although the W275A and F396A mutants showed only modest reductions in processivity, it was almost abolished by the W167A mutation. Thus, although aglycon subsites seem to steer processivity in ChiB, a glycon (-3) subsite seems to be adapted to do so in ChiA, in line with the notion that the two enzymes have different directionalities. Remarkably, whereas all three single mutants and the W167A/W275A double mutant showed reduced efficiency toward chitin, they showed up to 20-fold higher activities toward chitosan. These results show that the processive mechanism is essential for an efficient conversion of crystalline substrates but comes at a large cost in terms of intrinsic enzyme speed. This needs to be taken into account when devising enzymatic strategies for biomass turnover

Topics: Enzyme Catalysis and Regulation
Publisher: American Society for Biochemistry and Molecular Biology
OAI identifier: oai:pubmedcentral.nih.gov:2667748
Provided by: PubMed Central
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