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 acarviosyl moiety of acarbose to malto-oligosaccharides. This acarviosyl transferase (ATase) is encoded by a gene, acbD, in the putative biosynthetic gene cluster for the alpha-glucosidase inhibitor acarbose. The acbD gene was cloned and heterologously produced in Streptomyces lividans TK23. The recombinant protein was analyzed by enzyme assays. The AcbD protein (724 amino acids) displays all of the features of extracellular alpha-glucosidases and/or transglycosylases of the alpha-amylase family and exhibits the highest similarities 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 sequencing of tryptic peptides. AcbD had an apparent molecular mass of 76 kDa and an isoelectric point of 5.0 and required Ca(2+) ions for activity. The enzyme displayed maximal activity at 30 degrees C 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 donor and acceptor substrates were determined for the enzyme. Acceptors revealed a structural requirement for glucose-analogous structures conserving only the overall stereochemistry, except for the anomeric C atom, and the hydroxyl groups at positions 2, 3, and 4 of D-glucose. We discuss here the function of the enzyme in the extracellular formation of the series of acarbose-homologous compounds produced by Actinoplanes sp. strain SE50.
Hemker
M
M
Institut für Mikrobiologie, Westfälische Wilhelms-Universität, Correnstr. 3, D-48149 Münster, Germany.
Stratmann
A
A
Goeke
K
K
Schröder
W
W
Lenz
J
J
Piepersberg
W
W
Pape
H
H
D020909:Acarbose / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D015870:Gene Expression; D005798:Genes, Bacterial; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism*; D008848:Micromonosporaceae / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D016695:Glycosyltransferases; C432471:ACBD protein, Actinoplanes sp. 50-110; D020909:Acarbose