Polar residues lining the binding cleft of a Serratia marcescens family 18 chitinase position the substrate for attack and stabilize associative interactions

Abstract

Glycoside hydrolases (GHs) bind carbohydrates to convert polysaccharides into soluble sugars. GH binding sites are lined with aromatic and polar residues that facilitate function. The role of polar residues in GH function, relative to aromats, is often less straightforward. Using molecular dynamics simulation, thermodynamic integration and umbrella sampling molecular dynamics free energy calculations, and biochemical assays, we examined how Asp-316, Tyr-145, Arg-294, and Glu-221 aid in substrate binding, activity, and processive ability of a GH family 18 bacterial chitinase, Serratia marcescens chitinase B (ChiB). Despite what appears to be a ‘roof’ between Asp-316 and Trp-97 over the substrate in crystal structures, our results indicated ‘roof’ formation was not thermodynamically favourable. Asp-316 appeared to be supportive, providing interactions aiding in ligand stability at the −2 and −3 product sites. Mutation of Tyr-145 to alanine inactivated the enzyme; simulations revealed Tyr-145 stabilised the binding cleft strand containing the catalytic motif. Arg-294 appeared to play several roles, both stabilising Asp-215 and the +1 pyranose and forcing Trp-220 to stack with the +2 pyranose. Arg-294 may even modulate active site pK_a. Glu-221 hydrogen bonded with the +3 pyranose and appeared to stabilise the adjacent carbohydrate-π stacking between Phe-190 and the +3 pyranose.

GRAPHICAL ABSTRACT

KEYWORDS:

• Carbohydrate active enzyme (CAZy)
• chitin
• N-acetyl glucosamine
• chitosan
• processivity

Acknowledgements

The computational work primarily used the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation grant number ACI-1548562 under allocation number TG-MCB090159. Additional computational time was provided by University of Kentucky Center for Computational Sciences. This material is also based upon work supported by (while CMP is serving at) the NSF. Any opinion, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the NSF.

Disclosure statement

No potential conflict of interest was reported by the authors.

ORCID

Christina M. Payne http://orcid.org/0000-0001-5264-0964

Additional information

Funding

This work was supported by National Science Foundation: [grant number 1552355]; Norges Forskningsråd: [grant number 209335/F20].