The acbH gene of Actinoplanes sp. encodes a solute receptor with binding activities for acarbose and longer homologs.
Acarbose, a pseudomaltotetraose, is produced by strains of the genus Actinoplanes and is a potent inhibitor of alpha-glucosidases, including those from the human intestine. Therefore, it is used in the treatment of patients suffering from type 2 diabetes. The benefits of acarbose for the producer are not known; however, besides acting as an inhibitor of alpha-amylases secreted by competitors, a role as a 'carbophor' has been proposed. This would require a transport system mediating its uptake into the cytoplasm of Actinoplanes sp. A putative sugar ATP binding cassette (ABC) transport system, the genes of which are included within the biosynthetic gene cluster for acarbose, was suggested to be a possible candidate. The genes acbHFG encode a possible sugar binding protein (AcbH) and two membrane integral subunits (AcbFG). A gene coding for an ATPase component is missing. Since Actinoplanes sp. cannot yet be genetically manipulated we performed experiments to identify the substrate(s) of the putative transporter by assessing the substrate specificity of AcbH. The protein was overproduced in Escherichia coli as His10-fusion protein, purified under denaturating conditions and renatured. Refolding was verified by circular dichroism spectroscopy. Surface plasmon resonance studies revealed that AcbH binds acarbose and longer derivatives, but not maltodextrins, maltose or sucrose. Immunoblot analysis revealed the association of AcbH with the membrane fraction of Actinoplanes cells that were grown in the presence of maltose, maltodextrins or acarbose. Together, these findings suggest that the AcbHFG complex might be involved in the uptake of acarbose and are consistent with a role for acarbose as a 'carbophor'.
Brunkhorst
Claudia
C
Humboldt Universität zu Berlin, Institut für Biologie/Bakterienphysiologie, Chausseestr. 117, 10115 Berlin, Germany.
Wehmeier
Udo F
UF
Piepersberg
Wolfgang
W
Schneider
Erwin
E
D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism; D020909:Acarbose / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D008848:Micromonosporaceae / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D020909:Acarbose