Mutagenesis Study to Reveal Important Determinants of XXT2 Catalytic Activity

Xyloglucan (XyG) is the main hemicellulosic component of the primary cell wall in eudicots and nongraminaceous monocots. Three xyloglucan xylosyltransferases, including XXT2, are known to encode XyG xylosyltransferases, key enzymes in the process of XyG biosynthesis. A computational model of XXT2 was used to identify amino acids, F204, K207, D228, S229, D230, H378, putatively localized in the active site of XXT2 and mutagenesis study was performed to confirm predictions. All created mutants were expressed in E. coli SoluBL21 in pET20b plasmids containing an N‐terminal GB1 fusion tag. To increase yield and purity for future biophysical experiments, such as isothermal titration calorimetry and circular dichroism, the mutated genes were amplified from the pET20b plasmids, ligated into pGen2 plasmids and transfected into HEK293 cells. Results of the enzyme assay demonstrated that K207, D228, D230, and H378 are critical for enzyme activity due to their proposed coordination of the divalent cation. Reduced activity seen for the mutation made at F204 suggests that while this site is important for enzyme activity, it is not as crucial as the other sites tested. In addition, cysteine residues were selected for mutagenesis to investigate their effect on disulfide bond formation shown to be involved in hetero‐ and homo‐ dimer formations. Expression and analysis of these mutants is currently in process. Truncations of XXT2 from both the N‐ and C‐ends were used to examine the contributions of these termini to protein solubility and activity. In N‐terminal truncations longer than 31 residues, both protein expression and activity of XXT2 were negatively affected, indicating a possible effect on protein folding. Increasing sizes of truncations from the C‐terminus showed an increase in protein expression but a decrease in its catalytic activity. We conclude that the C‐terminus is not involved in protein folding but rather substrate binding. This truncation identified a series of six positively charged residues between 24 and 44 amino acids from the C‐terminus that may play a role in substrate binding. Mutagenesis of these residues revealed that four of the mutants have reduced catalytic activity. We conclude that the N‐terminal of XXT2 is involved in protein folding while the C‐terminal is involved in substrate binding. Furthermore, active site residues K207, D228, D230 and H378 are proved to be critical for enzymatic activity of XXT2. Support or Funding Information This research is supported by NSF‐MCB Grant #1121163