Molecular characterization of plant acidic alpha-mannosidase, a member of glycosylhydrolase family 38, involved in the turnover of N-glycans during tomato fruit ripening.
It has been reported that acidic α-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 α-mannosidase activity and fruit maturation at the molecular level, we have already purified and characterized an α-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 α-mannosidase gene using the yeast-expression system. The α-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 α-mannosidase, like the native enzyme, could cleave α1-2, 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 α-mannosidase could be superimposed on those of tomato α-mannosidase, suggesting that active site conformation is highly conserved between plant acidic α-mannosidase and animal lysosomal α-mannosidase.
Hossain
Md Anowar
MA
Department of Biofunctional Chemistry, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan.
Nakano
Ryohei
R
Nakamura
Kosuke
K
Hossain
Md Tofazzal
MT
Kimura
Yoshinobu
Y
D017485:1-Deoxynojirimycin / Q000494:pharmacology; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D005638:Fruit / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D018551:Solanum lycopersicum / Q000201:enzymology / Q000235:genetics; D008247:Lysosomes / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D011134:Polysaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D017026:Swainsonine / Q000494:pharmacology; D013696:Temperature; D043323:alpha-Mannosidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D006023:Glycoproteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D017485:1-Deoxynojirimycin; D043323:alpha-Mannosidase; D017026:Swainsonine