Identification, cloning, expression, and characterization of the extracellular acarbose-modifying glycosyltransferase, AcbD, from Actinoplanes sp strain SE50

An extracellular enzyme activity in the culture supernatant of the acarbose producer Actinoplanes sp, strain SE50 catalyzes the transfer of the acarvi osyl moiety of acarbose to malto-oligosaccharides. This acarviosyl transfer ase (ATase) is encoded by a gene, acbD, in the putative biosynthetic gene c luster for the alpha -glucosidase inhibitor acarbose, The acbD gene was clo ned and heterologously produced in Streptomyces lividans TK23. The recombin ant protein was analyzed by enzyme assays, The AcbD protein (724) amino aci ds) displays all of the features of extracellular alpha -glucosidases and/o r transglycosylases of the alpha -amylase family and exhibits the highest s imilarities to several cyclodextrin glucanotransferases (CGTases), However, AcbD had neither alpha -amylase nor CGTase activity. The AcbD protein was purified to homogeneity, and it was identified by partial protein sequencin g of tryptic peptides, AcbD had an apparent molecular mass of 76 kDa and an isoelectric point of 5.0 and required Ca2+ ions for activity. The enzyme d isplayed maximal activity at 30 degreesC and between pH 6.2 and 6.9. The K- m values of the ATase for acarbose (donor substrate) and maltose (acceptor substrate) are 0.65 and 0.96 mM, respectively. A wide range of additional d onor and acceptor substrates were determined for the enzyme. Accepters reve aled a structural requirement for glucose-analogous structures conserving o nly the overall stereochemistry, except for the anomeric C atom, and the hy droxyl groups at positions 2, 3, and 4 of D-glucose, We discuss here the fu nction of the enzyme in the extracellular formation of the series of acarbo se-homologous compounds produced by Actinoplanes sp, strain SE50.