Contributors: Chester
Context
Prior literature (per Alex) indicates GH10-domain exoglucanases can express beta-glucosidase activity, suggesting both the GH10 and GH1 domains of CxnA should be able to bind cellobiose.
GH10 domain — catalytic residues
Catalytic residues identified: GLU127 (proton donor) and GLU233 (catalytic nucleophile).
GLU127 mechanism: Assists bond-breaking during glycosylation; acts as base during deglycosylation (strips a proton from H₂O to release sugar). Normally sits in a conserved NEP motif — our GH10 sequence has NEA instead. Proline and alanine are chemically similar (non-polar, hydrophobic) despite structural differences; no steric hindrance identified, so NEA may function similarly to NEP. No literature precedent for NEA specifically was found.
GLU233 mechanism: Catalytic nucleophile — attacks the substrate’s carbon centre directly, forming a temporary covalent glycosyl-enzyme intermediate. Sits within the conserved ITEL motif, which is present in our sequence.
[Attachment: GH10.cif]
GH1 domain (UniProt: A0ACD6B8Z1)
Two conserved motifs identified and confirmed present: TFNEP and ITENG, each containing the relevant catalytic glutamate. Functions expected to parallel GH10 (proton donor in TFNEP; catalytic nucleophile in ITENG).
[Attachment: GH1.cif]
Cellobiose docking (preliminary)
Cellobiose docked into both GH1 and GH10 active sites via Autodock Vina to test for beta-glucosidase activity. Best-scoring poses (most negative ΔG) for both domains placed cellobiose near the relevant glutamates, with hydrogen-bond contacts observed.
Caveat: Docking proximity alone cannot prove catalytic activity — flagged for future wet-lab or dry-lab validation.