have led to the successful characterization of two key enzymes encoded by HP0159 (JHP0147) and HP1105 (JHP1032) open reading frames (ORFs) which are members of the large and diverse carbohydrate active enzymes (CAZY) GT-8 (-acetyl-glucosaminyl transferase.In addition, the activity screening approach led to the identification and characterization of a key core biosynthetic enzyme responsible for the biosynthesis of the α-1,6-glucan polymer.The acceptor/donor combinations were as follows: 1, fluorescein-5-EX succinimidyl ester-α-Man (FEX-α-Man) UDP-Glc NAc; 2, 6-(5-fluorescein-carboxamido)-hexanoic acid succinimidyl ester-Lac NAc (FCHASE-Lac NAc) UDP-Glc NAc; 3, FCHASE-β-Gal UDP-Glc; 4, FCHASE-α-Glc UDP-Glc; 5, FEX-α-Man UDP-Glc; 6, FCHASE-β-Glc NAc UDP-Gal; 7, FEX-α-Man UDP-Gal.The plus or minus signs indicate complete reactions or those missing the donor substrate, respectively. The acceptor/donor combinations were as follows: 1, fluorescein-5-EX succinimidyl ester-α-Man (FEX-α-Man) UDP-Glc NAc; 2, 6-(5-fluorescein-carboxamido)-hexanoic acid succinimidyl ester-Lac NAc (FCHASE-Lac NAc) UDP-Glc NAc; 3, FCHASE-β-Gal UDP-Glc; 4, FCHASE-α-Glc UDP-Glc; 5, FEX-α-Man UDP-Glc; 6, FCHASE-β-Glc NAc UDP-Gal; 7, FEX-α-Man UDP-Gal.Comparative analysis of this region in 26695 and J99 indicated that the JHP1032 and JHP1031 genes have likely arisen by gene duplication (60% identity), whereas in 26695 genome, there resides a single ORF (HP1105) which shows strong conservation at the protein level to both JHP1032 (73% identity) and JHP1031 (64% identity).To confirm that the JHP1032 ORF encodes the Glc NAc transferase activity, we subcloned the ORF into the expression vector p CW and measured enzyme activity following the induction of expression with isopropyl thio-b-D galactoside (IPTG) (data not shown).
Each pool was screened for β-1,4‐galactosyltransferase activity (UDP-Gal to FCHASE-Glc NAc) and for β--acetyl-glucosaminyltransferase activity [β-Glc NAc transferase (UDP-Glc NAc to FCHASE-Lac NAc)], and pools with β-Glc NAc transferase activity were plated for single colonies and rescreened to identify individual clones displaying the same activity.
Glycosyltransferases display remarkable diversity in their biosynthetic capacity, and it is this diversity which results in the production of a multitude of complex carbohydrates and polysaccharides which are recognized as key players in innumerable biological interactions (Allen and Kisailus, 1992).
To meet the challenge of the postgenomic era and to facilitate the functional characterization of the increasing number of sequences revealed in sequenced genomes to be involved in glycosidic bond formation, researchers have established an evolving hierarchical family classification scheme (Coutinho homologs) and define the role of each in LPS biosynthesis.
In the case of 26695, multiple product peaks were obvious by both TLC and CE analysis.
Assays with J99 lysates showed glucosyltransferase activity under the same reaction conditions, but only a single product was seen by TLC and CE.
Nuclear magnetic resonance (NMR) was used to confirm the linkage and determine that the Glc NAc was β-1,3 linked to Gal (see section The second strong enzymatic activity detected in the screens of 26695 and J99 lysates was that of glucosyltransferase activity.