ACARBOSE, A PSEUDOOLIGOSACCHARIDE, IS TRANSPORTED BUT NOT METABOLIZED BY THE MALTOSE-MALTODEXTRIN SYSTEM OF ESCHERICHIA COLI

The maltose regulon of Escherichia coli encompasses genes that are controlled by the positive regulator MalT and by cyclic AMP/CAP, the global regulator for carbon metabolism. Some of these genes are organized in clusters. The malA region at 76.5 min contains the malPQ operon encoding essential metabolic enzymes, maltodextrin phosphorylase and amylomaltase, respectively, and the divergently transcribed malT gene. Likewise, the malB region at 91.4 min contains the genes encoding the components of the transport system, organized in two divergently oriented operons: malEFG and malK lamB malM (22; for a recent review, see reference 6). With the exception of malT, the expression of these genes is induced when the MalT protein resides in the active conformation that is acquired by the simultaneous binding of maltotriose and ATP. Then, the protein binds to specific sites upstream of the respective promoters (MalT boxes), but transcription is not initiated unless cyclic AMP/CAP also binds upstream of the MalT boxes. This results in repositioning of MalT binding, thereby inducing the bending of the DNA, which eventually turns on transcription (24). The uptake of maltose and maltodextrins is accomplished by the combined action of five proteins: a specific channel protein in the outer membrane (maltoporin or LamB), a substrate-specific binding protein in the periplasm (MalE or maltose-binding protein), and a transport complex localized to the cytoplasmic membrane (MalFGK2) (reviewed in reference 6). The latter is a member of the superfamily of ATP-binding cassette transporter proteins (7, 15) and is composed of one copy each of the transmembrane proteins MalF and MalG and two copies of the ATP-hydrolyzing subunit MalK (9). While the crystal structures of maltoporin and MalE have been solved (25, 32), structural information on the membrane-bound complex is not yet available. However, crystals of the isolated MalK subunit from Salmonella typhimurium that diffract to a resolution of 3 Å were recently obtained (26). Maltodextrins and maltose (≤10 μM) cross the outer membrane through maltoporin molecules which are organized as homotrimers. Each subunit contains a channel that is formed by an 18-stranded, antiparallel β-barrel. Within the channels, the substrates are in contact with a “greasy slide” of aromatic residues that provide a path for translocation (12). In the periplasm, the ligands are readily complexed with maltose-binding protein that can exist in an open or a closed conformation, respectively. Binding of the substrate stabilizes the closed conformation (31). Only then can MalE productively interact with cytoplasmically exposed peptide loops of the membrane-integral components MalF and MalG. Through subsequent conformational changes of the latter, the presence of substrate is signaled, resulting in hydrolysis of ATP at the MalK subunits at the cytoplasmic side of the membrane. In turn, MalF and MalG are likely to be set into motion, and this leads to the substrate eventually being translocated across the membrane (10). In the cytoplasm, maltose and maltodextrins are attacked by the products of three genes, malQ, encoding an amylomaltase, malP, encoding a maltodextrin phosphorylase, and malZ, encoding a maltodextrin glucosidase. MalQ is essential for growth on maltose, while MalP is required for the utilization of maltodextrins only (6). Acarbose is a pseudooligosaccharide that is produced by strains of the genus Actinoplanes and is used to treat patients with diabetes. It is an effective inhibitor of α-amylases, glucosidases, and sucrases (36) and consists of an unsaturated aminocyclitol moiety (Fig. ​(Fig.1,1, ring A), a deoxyhexose (ring B) (together also called acarviosine), and a normal maltose (rings C and D). Prompted by its structural similarity to maltotetraose, we have studied the effects of acarbose on the metabolism of maltose and maltodextrins in whole cells of E. coli and on individual components of the maltose/maltodextrin system. Our results demonstrate that acarbose is efficiently transported but not metabolized by E. coli due to its poor performance as a substrate of maltodextrin-degrading enzymes. Also, the effects of acarbose on the channel properties of maltoporin were investigated in detail. FIG. 1 Structure of acarbose. The individual sugar residues are designated A to D (see the text for details).