Substrate specificity of the Escherichia coli maltodextrin transport system and its component proteins

Maltooligosaccharides up to maltoheptaose are transported by the maltodextrin transport system of Escherichia coli. The overall substrate specificity of the transport system was investigated by using 15 maltodextrin analogues with various modifications at the reducing end of the oligosaccharides as competing substrates. The binding interaction of the analogues with maltoporin in the outer membrane and the periplasmic maltose-binding protein, the two protein components of the transport system with known specificity for maltodextrins, was also investigated. All analogues containing several α,1 → 4-glucosyl linkages were bound with high affinity by maltoporin and maltose-binding protein, regardless of O-methyl, O-nitrophenyl, β-glucosyl or β-fructosyl substitutions at the reducing end of the dextrins. Introduction of a negative charge or lack of a ring structure at the reducing end were also ineffective in abolishing binding by these two proteins. These results suggest that the structure of the reducing glucose is not important in the binding specificity of maltoporin or maltose-binding protein. However, the high affinity of these proteins for analogues was not in itself sufficient for recognition by the transport system overall. Maltohexaitol, 4-nitrophenyl α-maltotetraoside and 4-β-d-maltopentaosyl-d-glucopyranose were bound with the same affinity as comparable maltodextrins by both maltoporin and maltose-binding protein but were poorly recognized by the transport system. These results suggest that another, yet uninvestigated component of the transport system has a more restricted specificity towards changes at the reducing end of the maltodextrin molecule.     

The maltodextrin system of Escherichia coli: glycogen-derived endogenous induction and osmoregulation

Strains of Escherichia coli lacking MalQ (maltodextrin glucanotransferase or amylomaltase) are endogenously induced for the maltose regulon by maltotriose that is derived from the degradation of glycogen (glycogen-dependent endogenous induction). A high level of induction was dependent on the presence of MalP, maltodextrin phosphorylase, while expression was counteracted by MalZ, maltodextrin glucosidase. Glycogen-derived endogenous induction was sensitive to high osmolarity. This osmodependence was caused by MalZ. malZ, the gene encoding this enzyme, was found to be induced by high osmolarity even in the absence of MalT, the central regulator of all mal genes. The osmodependent expression of malZ was neither RpoS nor OmpR dependent. In contrast, the malPQ operon, whose expression was also increased at a high osmolarity, was partially dependent on RpoS. In the absence of glycogen, residual endogenous induction of the mal genes that is sensitive to increasing osmolarity can still be observed. This glycogen-independent endogenous induction is not understood, and it is not affected by altering the expression of MalP, MalQ, and MalZ. In particular, its independence from MalZ suggests that the responsible inducer is not maltotriose.     

Chaperone-assisted refolding of Escherichia coli maltodextrin glucosidase

In vitro refolding of maltodextrin glucosidase, a 69 kDa monomeric Escherichia coli protein, was studied in the presence of glycerol, dimethylsulfoxide, trimethylamine-N-oxide, ethylene glycol, trehalose, proline and chaperonins GroEL and GroES. Different osmolytes, namely proline, glycerol, trimethylamine-N-oxide and dimethylsulfoxide, also known as chemical chaperones, assist in protein folding through effective inhibition of the aggregation process. In the present study, it was observed that a few chemical chaperones effectively reduced the aggregation process of maltodextrin glucosidase and hence the in vitro refolding was substantially enhanced, with ethylene glycol being the exception. Although, the highest recovery of active maltodextrin glucosidase was achieved through the ATP-mediated GroEL/GroES-assisted refolding of denatured protein, the yield of correctly folded protein from glycerol- or proline-assisted spontaneous refolding process was closer to the chaperonin-assisted refolding. It was also observed that the combined application of chemical chaperones and molecular chaperone was more productive than their individual contribution towards the in vitro refolding of maltodextrin glucosidase. The chemical chaperones, except ethylene glycol, were found to provide different degrees of protection to maltodextrin glucosidase from thermal denaturation, whereas proline caused the highest protection. The observations from the present studies conclusively demonstrate that chemical or molecular chaperones, or the combination of both chaperones, could be used in the efficient refolding of recombinant E. coli maltodextrin glucosidase, which enhances the possibility of identifying or designing suitable small molecules that can act as chemical chaperones in the efficient refolding of various aggregate-prone proteins of commercial and medical importance.     

Trehalose transport and metabolism in Escherichia coli.

Trehalose metabolism in Escherichia coli is complicated by the fact that cells grown at high osmolarity synthesize internal trehalose as an osmoprotectant, independent of the carbon source, although trehalose can serve as a carbon source at both high and low osmolarity. The elucidation of the pathway of trehalose metabolism was facilitated by the isolation of mutants defective in the genes encoding transport proteins and degradative enzymes. The analysis of the phenotypes of these mutants and of the reactions catalyzed by the enzymes in vitro allowed the formulation of the degradative pathway at low osmolarity. Thus, trehalose utilization begins with phosphotransferase (IITre/IIIGlc)-mediated uptake delivering trehalose-6-phosphate to the cytoplasm. It continues with hydrolysis to trehalose and proceeds by splitting trehalose, releasing one glucose residue with the simultaneous transfer of the other to a polysaccharide acceptor. The enzyme catalyzing this reaction was named amylotrehalase. Amylotrehalase and EIITre were induced by trehalose in the medium but not at high osmolarity. treC and treB encoding these two enzymes mapped at 96.5 min on the E. coli linkage map but were not located in the same operon. Use of a mutation in trehalose-6-phosphate phosphatase allowed demonstration of the phosphoenolpyruvate- and IITre-dependent in vitro phosphorylation of trehalose. The phenotype of this mutant indicated that trehalose-6-phosphate is the effective in vivo inducer of the system.     

The crystal structure of the Escherichia coli maltodextrin phosphorylase-acarbose complex

Acarbose is a naturally occurring pseudo-tetrasaccharide. It has been used in conjunction with other drugs in the treatment of diabetes where it acts as an inhibitor of intestinal glucosidases. To probe the interactions of acarbose with other carbohydrate recognition enzymes, the crystal structure of E. coli maltodextrin phosphorylase (MalP) complexed with acarbose has been determined at 2.95 A resolution and refined to crystallographic R-values of R (Rfree) = 0.241 (0.293), respectively. Acarbose adopts a conformation that is close to its major minimum free energy conformation in the MalP-acarbose structure. The acarviosine moiety of acarbose occupies sub-sites +1 and +2 and the disaccharide sub-sites +3 and +4. (The site of phosphorolysis is between sub-sites -1 and +1.) This is the first identification of sub-sites +3 and +4 of MalP. Interactions of the glucosyl residues in sub-sites +2 and +4 are dominated by carbohydrate stacking interactions with tyrosine residues. These tyrosines (Tyr280 and Tyr613, respectively, in the rabbit muscle phosphorylase numbering scheme) are conserved in all species of phosphorylase. A glycerol molecule from the cryoprotectant occupies sub-site -1. The identification of four oligosaccharide sub-sites, that extend from the interior of the phosphorylase close to the catalytic site to the exterior surface of MalP, provides a structural rationalization of the substrate selectivity of MalP for a pentasaccharide substrate. Crystallographic binding studies of acarbose with amylases, glucoamylases, and glycosyltranferases and NMR studies of acarbose in solution have shown that acarbose can adopt two different conformations. This flexibility allows acarbose to target a number of different enzymes. The two alternative conformations of acarbose when bound to different carbohydrate enzymes are discussed.     

Enzymatic catalysis in crystals of Escherichia coli maltodextrin phosphorylase

The bacterial enzyme maltodextrin phosphorylase (MalP) catalyses the phosphorolysis of an alpha-1,4-glycosidic bond in maltodextrins, removing the non-reducing glucosyl residues of linear oligosaccharides as glucose-1-phosphate (Glc1P). In contrast to the well-studied muscle glycogen phosphorylase (GP), MalP exhibits no allosteric properties and has a higher affinity for linear oligosaccharides than GP. We have used MalP as a model system to study catalysis in the crystal in the direction of maltodextrin synthesis. The 2.0A crystal structure of the MalP/Glc1P binary complex shows that the Glc1P substrate adopts a conformation seen previously with both inactive and active forms of mammalian GP, with the phosphate group not in close contact with the 5'-phosphate group of the essential pyridoxal phosphate (PLP) cofactor. In the active MalP enzyme, the residue Arg569 stabilizes the negative-charged Glc1P, whereas in the inactive form of GP this key residue is held away from the catalytic site by loop 280s and an allosteric transition of the mammalian enzyme is required for activation. The comparison between MalP structures shows that His377, through a hydrogen bond with the 6-hydroxyl group of Glc1P substrate, triggers a conformational change of the 380s loop. This mobile region folds over the catalytic site and contributes to the specific recognition of the oligosaccharide and to the synergism between substrates in promoting the formation of the MalP ternary complex. The structures solved after the diffusion of oligosaccharides (either maltotetraose, G4 or maltopentaose, G5) into MalP/Glc1P crystals show the formation of phosphate and elongation of the oligosaccharide chain. These structures, refined at 1.8A and at 2.2A, confirm that only when an oligosaccharide is bound to the catalytic site will Glc1P bend its phosphate group down so it can contact the PLP 5' phosphate group and promote catalysis. The relatively large oligosaccharide substrates can diffuse quickly into the MalP/Glc1P crystals and the enzymatic reaction can occur without significant crystal damage. These structures obtained before and after catalysis have been used as frames of a molecular movie. This movie reveals the relative positions of substrates in the catalytic channel and shows a minimal movement of the protein, involving mainly Arg569, which tracks the substrate phosphate group.     

The gusBC genes of Escherichia coli encode a glucuronide transport system

Two genes, gusB and gusC, from a natural fecal isolate of Escherichia coli are shown to encode proteins responsible for transport of beta-glucuronides with synthetic [(14)C]phenyl-1-thio-beta-d-glucuronide as the substrate. These genes are located in the gus operon downstream of the gusA gene on the E. coli genome, and their expression is induced by a variety of beta-d-glucuronides. Measurements of transport in right-side-out subcellular vesicles show the system has the characteristics of secondary active transport energized by the respiration-generated proton motive force. When the genes were cloned together downstream of the tac operator-promoter in the plasmid pTTQ18 expression vector, transport activity was increased considerably with isopropylthiogalactopyranoside as the inducer. Amplified expression of the GusB and GusC proteins enabled visualization and identification by N-terminal sequencing of both proteins, which migrated at ca. 32 kDa and 44 kDa, respectively. Separate expression of the GusB protein showed that it is essential for glucuronide transport and is located in the inner membrane, while the GusC protein does not catalyze transport but assists in an as yet unknown manner and is located in the outer membrane. The output of glucuronides as waste by mammals and uptake for nutrition by gut bacteria or reabsorption by the mammalian host is discussed.     

D-serine transport system in Escherichia coli K-12

The d-serine transport system in Escherichia coli K-12 was studied by use of a mutant unable to form d-serine deaminase, yet resistant to d-serine. The mutant is greatly impaired in its ability to accumulate d-serine, d-alanine, and glycine. Transport of l-alanine is partially affected but transport of l-serine is unaffected. The mutant is also resistant to d-cycloserine, indicating that d-serine is transported by the system responsible for uptake of d-cycloserine. The d-serine transport system is not inducible, but appears to be formed constitutively, as are the transport systems of most amino acids. The transport mutation appears to be multistep and maps to the right of malB on the E. coli linkage map.     

Specialized peptide transport system in Escherichia coli.

Trileucine is utilized as a source of leucine for growth of strains of Escherichia coli K-12 that are deficient in the oligopeptide transport system (Opp). Trithreonine is toxic to E. coli K-12. Opp- mutants of E. coli K-12 retain complete sensitivity to this tripeptide. Moreover, E. coli W, which is resistant to trithreonine, can utlize this tripeptide as a threonine source and this capability is fully maintained in E. coli W (Opp-). A spontaneous trithreonine-resistant mutant of E. coli K-12 (Opp-) has been isolated that has an impaired growth response to trileucine and is resistant to trithreonine. Trileucine competes with the uptake of trithreonine as measured by its ability to relieve trithreonine toxicity in E. coli K-12. It is concluded that trileucine as well as trithreonine are transported into E. coli K-12 or W by a common uptake system that is distinct from the Opp system. Trimethionine can act as a competitor of trileucine or trithreonine-supported growth and as an antagonist of trithreonine toxicity in Opp- mutants. It is concluded that trimethionine is recognized by the trileucine-trithreonine transport system. Trithreonine, trimethionine, and trileucine are also transported by the Opp system, as they all relieve triornithine toxicity towards E. coli W and compete with tetralysine utilization as lysine source for growth of a lysine auxotroph of this strain.     

Specificity of the Escherichia coli proline transport system.

The presence of both the carbonyl portion of the carboxyl group at position 2 of the pyrrolidine ring and a secondary amine was essential for uptake of a compound by the proline permease of Escherichia coli. The permease possessed a high affinity for azetidine-2-carboxylic acid and for compounds with ring structures smaller than the pyrrolidine ring. Pipecolic acid, the higher homologue of proline, and its derivatives were not transported. Cis- and trans-3,4-methano-prolines, also six-membered ring structures, behaved anomolously in that they possessed a high affinity for the permease. The difference between the methano-prolines and other six-membered ring structures probably resides in the fact that the former exist in the "boat" configuration whereas the latter possess the "chair" configuration. In general, substituted prolines in the cis configuration displayed a higher affinity for the permease than did corresponding trans isomers, though the affinity for substituted prolines was influenced by the position, size, and polar or nonpolar nature of the substituent group. At O C many analogues with affinity for proline permease exchanged with intracellular proline, but some analogues, notably trans-3-methyl- and trans-4-methyl-L-prolines, though possessing high affinity for the permease, showed an almost complete inability to exchange with intracellular proline.     

Phosphate-specific transport system of Escherichia coli: nucleotide sequence and gene-polypeptide relationships.

The DNA nucleotide sequence of four genes for the phosphate-specific transport system of Escherichia coli is reported. Along with the DNA sequence for the phoS gene reported previously (Surin et al., J. Bacteriol. 157:772-778, 1984; Magota et al., J. Bacteriol. 157:909-917, 1984), this study completes the nucleotide sequence of the phosphate-specific transport region. The complete sequence (including phoS) contains five open reading frames oriented in the same direction, each preceded by a putative ribosome-binding site near the presumed translation initiation codon ATG. The complete sequence is transcribed counterclockwise, in the order phoS pstC pstA pstB phoU. Genetic complementation shows that of the four open reading frames in the new sequence, three correspond to known mutant alleles; the fourth, which was designated pstC, has not been described before and could not be related to any known mutant allele. We have confirmed that pstA was allelic to phoT32. The pstC, pstB, and phoU gene products were identified as peripheral membrane proteins. The pstA gene product appears to be an integral membrane protein.     

The metabolism of isocytidine in Escherichia coli

Intact cells and cell-free extracts of E. coli convert isocytidine to isocytosine and uracil. The radioactive label of 5-[³H]isocytidine is incorporated into RNA and, DNA of growing bacteria at a rate equal to about 1.4% of that of cytidine under similar conditions; the radioactivity is found in uridylic, cytidylic and 2′-deoxythymidylic acids, while less than 0.4% of incorporated radioactive material might be due to possible incorporation of intact isocytidine. Uridine phosphorylase and cytidine deaminase apparently do not participate in the metabolic conversion of isocytidine.     

Citrate-dependent iron transport system in Escherichia coli K-12

Induction of the citrate-dependent iron transport system of Escherichia coli K-12 required 0.1 mM citrate and 0.1 micrometer iron in the growth medium. Five--ten-times more iron than citrate was taken up into the cells which suggests that citrate was largely excluded from the transport. Fluorocitrate and phosphocitrate induced the citrate-dependent iron transport system although they supported iron uptake only very poorly. An outer membrane protein (FecA), belonging to the transport system, was induced in fecB mutants which were devoid of citrate-dependent iron transport. The intracellular citrate and iron concentrations were 10--100-times higher than the external concentrations required for induction of the transport system. It is concluded that only exogenous ferric citrate induced the transport system, and that citrate did not have to enter the cytoplasm. The Tn10 transposon, conferring tetracycline resistance, was inserted near the fec gene region which controls the expression of the citrate-dependent iron transport system. The determination of the cotransduction frequencies of Tn10 with the fecA and fecB markers suggested the gene order fecA fecB Tn10.