Engineering of substrate specificity in a plant cell-wall modifying enzyme through alterations of carboxyl-terminal amino acid residues

Abstract

Structural determinants of substrate recognition remain inadequately defined in broad specific cell-wall modifying enzymes, termed xyloglucan xyloglucosyl transferases (XETs). Here, we investigate the Tropaeolum majus seed TmXET6.3 isoform, a member of the GH16_20 subfamily of the GH16 network. This enzyme recognises xyloglucan (XG)-derived donors and acceptors, and a wide spectrum of other chiefly saccharide substrates, although it lacks the activity with homogalacturonan (pectin) fragments. We focus on defining the functionality of carboxyl-terminal residues in TmXET6.3, which extend acceptor binding regions in the GH16_20 subfamily but are absent in the related GH16_21 subfamily. Site-directed mutagenesis using double to quintuple mutants in the carboxyl-terminal region - substitutions emulated on barley XETs recognising the XG/penta-galacturonide acceptor substrate pair - demonstrated that this activity could be gained in TmXET6.3. We demonstrate the roles of semi-conserved Arg238 and Lys237 residues, introducing a net positive charge in the carboxyl-terminal region (which complements a negative charge of the acidic penta-galacturonide) for the transfer of xyloglucan fragments. Experimental data, supported by molecular modelling of TmXET6.3 with the XG oligosaccharide donor and penta-galacturonide acceptor substrates, indicated that they could be accommodated in the active site. Our findings support the conclusion on the significance of positively charged residues at the carboxyl terminus of TmXET6.3 and suggest that a broad specificity could be engineered via modifications of an acceptor binding site. The definition of substrate specificity in XETs should prove invaluable for defining the structure, dynamics, and function of plant cell walls, and their metabolism; these data could be applicable in various biotechnologies.

Keywords: computational chemistry; docking and molecular dynamics; donor and acceptor substrates; electrostatic potential; hetero-transglycosylation; pectin fragment; site-directed mutagenesis.