Unusual Β1-4-galactosidase Activity of an Α1-6-mannosidase from Xanthomonas Manihotis in the Processing of Branched Hybrid and Complex Glycans

Mannosidases are a diverse group of glycoside hydrolases that play crucial roles in mannose trimming of oligomannose glycans, glycoconjugates, and glycoproteins involved in numerous cellular processes, such as glycan biosynthesis and metabolism, structure regulation, cellular recognition, and cell-pathogen interactions. Exomannosidases and endomannosidases cleave specific glycosidic bonds of mannoside linkages in glycans and can be used in enzyme-based methods for sequencing of isomeric glycan structures. alpha 1-6-mannosidase from Xanthomonas manihotis is known as a highly specific exoglycosidase that removes unbranched alpha 1-6 linked mannose residues from oligosaccharides. However, we discovered that this alpha 1-6-mannosidase also possesses an unexpected beta 1-4-galactosidase activity in the processing of branched hybrid and complex glycans through our use of enzymatic reactions, high performance anion-exchange chromatography, and liquid chromatography mass spectrometric sequencing. Our docking simulation of the alpha 1-6-mannosidase with glycan substrates reveals potential interacting residues in a relatively shallow pocket slightly differing from its homologous enzymes in the glycoside hydrolase 125 family, which may be responsible for the observed higher promiscuity in substrate binding and subsequent terminal glycan hydrolysis. This observation of novel beta 1-4-galactosidase activity of the alpha 1-6-mannosidase provides unique insights into its bifunctional activity on the substrate structure-dependent processing of terminal alpha 1-6-mannose of unbranched glycans and terminal beta 1-4-galactose of hybrid and complex glycans. The finding thus suggests the dual glycosidase specificity of this alpha 1-6-mannosidase and the need for careful consideration when used for the structural elucidation of glycan isomers.