Substrate recognition domains as revealed by active hybrids between the D-arabinitol and ribitol transporters from Klebsiella pneumoniae

Abstract

Two new genes, dalT and rbtT, have been cloned from the dal operon for D-arabinitol and the rbt operon for ribitol uptake and degradation, respectively, in Klebsiella pneumoniae 1033-5P14, derivative KAY2026. Each gene codes for a specific transporter which, based on sequence data, belongs to a large family of carbohydrate transporters which constitutes 12 transmembrane helices. DalT and RbtT show an unusually high similarity (86.2% identical residues for totals of 425 and 427 amino acids, respectively). This allowed the construction of DalT-RbtT and RbtT-DalT crossover hybrids by using a natural restriction site overlapping Met202. This site is located within the large cytoplasmic loop which connects the putative helices 6 and 7 and in particular the amino-and the carboxy-terminal halves of the transporters. Both hybrids have close to normal transport activities but essentially the substrate specificities and kinetic properties of the amino-terminal half. This result localizes essential substrate binding and recognition sites to the amino-terminal halves of the proteins in this important class of carbohydrate transporters.Membrane transport systems from prokaryotic and eukaryotic organisms form a large superfamily of facilitators (MFS) that catalyzes uniport, symport, and antiport (11,19). Members of the MFS superfamily share conserved amino acid sequence motifs and structures. These transporters include in particular facilitators and H ϩ -symporters for pentoses, hexoses, and polyhydric alcohols. The presence of conserved sequences and structural similarities implies common mechanisms of action. Replacement by localized mutagenesis of defined amino acids which impair transport properties has been used to identify putative catalytic centers. Alternatively, hybrids have been constructed in which a part of a transporter is replaced by the corresponding part of a related transporter having, e.g., different substrate specificity. At present, active hybrids only have been obtained when small parts have been exchanged or when very closely related transporters, as often found in one eukaryotic organism, have been used, e.g., four human glucose transporters (4, 35) or the glucose and galactose transporters from yeast (23). The results from such studies with 12 transmembrane helix facilitators and H ϩ -symporters tend to localize a region involved in substrate recognition to the carboxy-terminal half or they indicate the presence of a second site in the middle part of these transporters.In this paper we describe hybrids between a transporter (DalT) for the polyhydric alcohol (pentitol) D-arabinitol and one (RbtT) for its isomer ribitol. The corresponding genes, dalT and rbtT, respectively, have been located within the dal and the rbt operons of Klebsiella pneumoniae 1033-5P14 and have been cloned from the chromosome of its derivative KAY2026. Direct (15) and indirect (9,13,21,27,30,34) evidence for the existence of a transporter for each pentitol in this organism has been given. The corresponding genes, however, have no...

Evolutionary Dynamics of the Yersinia enterocolitica Complex

“…A similar, conserved D-arabitol operon structure has been described in Klebsiella pneumoniae and function confirmed (Heuel, Turgut, Schmid and Lengeler, 1997). exclusive to non-pathogenic BTs though (Fantasia Mazzotti, et al, 1985;Fantasia Mazzotti, Mingrone and Martini, 1987), as some BT 1A strains can be negative, just as some pathogenic BT 3 strains were found to metabolize D-arabitol.…”

Arabitol Metabolism of Corynebacterium glutamicum and Its Regulation by AtlR

“…Growth on arabitol has been described for several microorganisms, e.g., for Enterobacter aerogenes (15,16,76,77), K. pneumoniae (34,35), Rhizobium meliloti and Rhizobium trifolii (53,62), E. coli C strains (64), and Pseudomonas fluorescens (13). All these organisms possess D-AraDH activity, mediated by NAD ϩ -dependent dehydrogenases (either AraDH or ManDH), and oxidize arabitol to D-xylulose, which is then phosphorylated by a XylK protein.…”

“…For arabitol utilization by C. glutamicum, here we present evidence for the involvement of a sugar alcohol permease (encoded by rbtT), an enzyme with AraDH and ManDH activities (encoded by mtlD), XylK (encoded by xylB), and AtlR (encoded by atlR), which was previously identified as a regulator for some genes encoding enzymes of the central metabolism in C. glutamicum (see the introduction). The rbtT gene product shows significant identity (29%) to an H ϩ symporter (DalT) of K. pneumoniae, belonging to the major facilitator superfamily (MFS) of carbohydrate transporters and being responsible for arabitol uptake in this organism (35). The mtlD gene product shows a striking level of identity (up to 68%) with bacterial ManDHs, which, at least in some cases, have also been shown to catalyze arabitol oxidation with NAD ϩ (e.g., see references 14, 15, and 72).…”

Mutations Which Uncouple Transport and Phosphorylation in the d -Mannitol Phosphotransferase System of Escherichia coli K-12 and Klebsiella pneumoniae 1033-5P14

“…Transport, binding, phosphorylation, and enzyme assays. Transport of 14 Clabeled D-mannitol, D-glucitol, or D-arabinitol (usually at a final concentration of 10 M) was tested by using exponentially growing cells, and the activities were calculated from the initial uptake rates (0 to 30 s) as described previously (10,44). The tests used for the D-mannitol 1-phosphate dehydrogenase (MtlD) and for the D-arabinitol dehydrogenase (DalD) have also been described previously in detail (15,28).…”