Molecular characterization of plant acidic α-mannosidase, a member of glycosylhydrolase family 38, involved in the turnover of N-glycans during tomato fruit ripening

It has been reported that acidic alpha-mannosidase activity increases during tomato fruit ripening, suggesting the turnover of N-glycoproteins is deeply associated with fruit ripening. As part of a study to reveal the relationship between the plant alpha-mannosidase activity and fruit maturation at the molecular level, we have already purified and characterized an alpha-mannosidase from tomato fruit (Hossain et al., Biosci. Biotechnol. Biochem. 2009; 73: 140-146). In this article, we describe the identification and expression of the tomato acidic alpha-mannosidase gene using the yeast-expression system. The alpha-mannosidase-gene located at chomosome 6 is a 10 kb spanned containing 30 exons. The gene-encoded-protein is single polypeptide chain of 1,028 amino acids containing glycosyl hydrolase domain-38 with predicted molecular mass of 116 kDa. The recombinant enzyme showed maximum activity at pH 5.5, and was almost completely inhibited by both of 1-deoxymannojirimycin and swainsonine. The recombinant alpha-mannosidase, like the native enzyme, could cleave alpha 1-2, 1-3 and 1-6 mannosidic linkage from both high-mannose and truncated complex-type N-glycans. A molecular 3D modelling shows that catalytically important residues of animal lysosomal alpha-mannosidase could be superimposed on those of tomato alpha-mannosidase, suggesting that active site conformation is highly conserved between plant acidic alpha-mannosidase and animal lysosomal alpha-mannosidase.