Identification, Cloning, Expression, and Characterization of the Extracellular Acarbose-Modifying Glycosyltransferase, AcbD, fromActinoplanessp. Strain SE50

Abstract

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 ␣-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 ␣-glucosidases and/or transglycosylases of the ␣-amylase family and exhibits the highest similarities to several cyclodextrin glucanotransferases (CGTases). However, AcbD had neither ␣-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°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.

Biosynthesis of the C7-cyclitol Moiety of Acarbose inActinoplanes Species SE50/110

“…SE50/110 has been cloned on a single cosmid and fully sequenced recently. 2 Its function was proven by heterologous expression from the cosmid in S. lividans TK23, which formed acarbose-related substances and characteristic enzyme activities involved in acarbose metabolism such as the extracellular acarviosyltransferase AcbD (28), and the cytoplasmic acarbose 7-kinase (18,19). The gene for the acarbose 7-kinase, acbK, was identified in a putative operon, acbKLM-NOC (GenBank TM /EBI accession number Y18523) (Fig.…”

Enzymatic Synthesis of Acarviosyl-maltooligosaccharides Using Disproportionating Enzyme 1

“…strain SE50, transfers the acarviosyl unit from acarbose to various acceptors, including maltooligosaccharides, xylobiose, and laminaribiose. 6) Maltogenic amylase from Bacillus stearothermophilus acts on acarbose and produces acarviosyl-glucose by hydrolysis and acarviosyl-isomaltose by transglycosylation of the acarviosyl-glucose unit to glucose. 7) Cyclomaltodextrin glucanotransferase from B. macerans and dextransucrases from Leuconostoc mesenteroides use acarbose as acceptor and attach the maltohexaose of cyclomaltohexaose and the glucose of sucrose respectively to the reducing-or nonreducing-end of acarbose.…”

Single Amino Acid Mutations Interchange the Reaction Specificities of Cyclodextrin Glycosyltransferase and the Acarbose-Modifying Enzyme Acarviosyl Transferase

“…SE50/110 that transfers the acarviosyl moiety of acarbose to the 4-hydroxyl group of various sugars (14) (Figure 1). This is the only enzyme known to catalyze this reaction.…”

“…This is the only enzyme known to catalyze this reaction. ATase has the highest sequence identity (42%) with CGTases (14), and the protein is most likely organized in five domains (A-E), similar to CGTases. The substrate of ATase, acarbose, is a pseudotetrasaccharide, composed of the C7-cyclitol valienamine, 4-amino-4,6-dideoxyglucose, and maltose ( Figure 3), that is found in culture medium from Actinoplanes sp.…”

“…SE50/110 (26,27). Despite its high similarity to CGTases, it has been reported that ATase has no cyclodextrin-forming activity and is not inhibited by acarbose (14). Rather, acarbose is its main substrate, with a K M value of 0.65 mM (14).…”