Effect of Gln469 on the Activity and Substrate Specificity of the N-glycosyltransferase from Actinobacillus pleuropneumoniae

N-Glycosyltransferase from Actinobacillus pleuropneumoniae (ApNGT) belongs to a novel class of N-glycosylation-catalyzing enzymes and has been extensively studied in decade. The enzyme transfers UDP-Glc/Gal to the asparagine side chains of the receptor proteins/peptides bearing Asn-Xaa-Ser/Thr (where Xaa ≠ Pro) sequons. Recently, the residue of Gln469 located on a unique loop within the active site has been demonstrated to contribute to the stringent selectivity of ApNGT. The relaxed acceptor specificity has been achieved by replacing Gln469 with small residue (such as Ala). To obtain more flexible N-glycosylation biocatalysts for use in glycoengineering applications and also in-depth understanding of the role of Gln469 in catalysis and substrate binding, a set of mutants with deletions of Gln469 and/or its neighboring residues were herein constructed by overlap extension PCR. The activity and specificity of ApNGT were systematically investigated by screening 3 panels of synthetic peptide substrates. Collectively, the results indicated that Gln469 may engage in amino acid residue selectivity and is not requisite for catalysis. Meanwhile, the Δ469 mutant reveals relaxed N-glycosylation site selection and is capable of efficiently glycosylating peptide with positive charged Lys/Arg at +1 position that, however, is disfavored by the wild type ApNGT and other known microbial N-glycosyltransferases.