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Download fileAromatic-Mediated Carbohydrate Recognition in Processive Serratia marcescens Chitinases
journal contribution
posted on 2016-01-29, 00:00 authored by Suvamay Jana, Anne Grethe Hamre, Patricia Wildberger, Matilde
Mengkrog Holen, Vincent G. H. Eijsink, Gregg T. Beckham, Morten Sørlie, Christina M. PayneMicroorganisms use a host of enzymes,
including processive glycoside
hydrolases, to deconstruct recalcitrant polysaccharides to sugars.
Processive glycoside hydrolases closely associate with polymer chains
and repeatedly cleave glycosidic linkages without dissociating from
the crystalline surface after each hydrolytic step; they are typically
the most abundant enzymes in both natural secretomes and industrial
cocktails by virtue of their significant hydrolytic potential. The
ubiquity of aromatic residues lining the enzyme catalytic tunnels
and clefts is a notable feature of processive glycoside hydrolases.
We hypothesized that these aromatic residues have uniquely defined
roles, such as substrate chain acquisition and binding in the catalytic
tunnel, that are defined by their local environment and position relative
to the substrate and the catalytic center. Here, we investigated this
hypothesis with variants of Serratia marcescens family
18 processive chitinases ChiA and ChiB. We applied molecular simulation
and free energy calculations to assess active site dynamics and ligand
binding free energies. Isothermal titration calorimetry provided further
insight into enthalpic and entropic contributions to ligand binding
free energy. Thus, the roles of six aromatic residues, Trp-167, Trp-275,
and Phe-396 in ChiA, and Trp-97, Trp-220, and Phe-190 in ChiB, have
been examined. We observed that point mutation of the tryptophan residues
to alanine results in unfavorable changes in the free energy of binding
relative to wild-type. The most drastic effects were observed for
residues positioned at the “entrances” of the deep substrate-binding
clefts and known to be important for processivity. Interestingly,
phenylalanine mutations in ChiA and ChiB had little to no effect on
chito-oligomer binding, in accordance with the limited effects of
their removal on chitinase functionality.
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Processive glycoside hydrolasesenzymeTrphydrolyticsubstrate chain acquisitionresidueligand bindingmutationprocessive glycoside hydrolasesSerratia marcescens family 18 processive chitinases ChiAChiBIsothermal titration calorimetrytunnelProcessive Serratia marcescens ChitinasesMicroorganisms usePherolecleft