Polyamine Transport in E coli

E. coli contain putrescine and spermidine. In addition, cadaverine and aminopropyl-cadaverine can function similarly to putrescine and spermidine (11). Therefore, we first tried to identify the genes encoding the transport proteins for putrescine, spermi-dine, and cadaverine. To identify these genes, a mutant that is deficient in polyamine transport was first isolated. Genes for polyamine transport systems were then isolated by transforming the mutant with DNA fragments using pACYC184 as a vector. One clone for the genes encoding proteins catalyzing both putrescine and spermidine uptake (pPT104) was isolated. Two clones for the genes of proteins catalyzing only putrescine uptake (pPT79 and pPT71) were obtained (12). The Km values for spermidine and putrescine of the polyamine transport system encoded by pPT104 were 0.1 and 1.5 |iM, respectively. The Km value for putrescine of the putrescine transport systems

From: Polyamine Cell Signaling: Physiology Pharmacology and Cancer Research Edited by: J.-Y. Wang and R. A. Casero, Jr. © Humana Press Inc., Totowa, NJ

Spermidine-preferential Putrescine-specific Putrescine Cadaverine uptake system uptake system transporter transporter

Spermidine-preferential Putrescine-specific Putrescine Cadaverine uptake system uptake system transporter transporter

Polyamine Transport System
Fig. 1. Polyamine transport systems in Escherichia coli.

encoded by pPT79 and pPT71 was 0.5 and 1.8 |iM,, respectively. Subsequently, cadB encoding the cadaverine transporter was identified (13,14).

Polyamine transporters are classified into two groups: one consists of ABC (ATP-binding cassette) transporters encoded by pPT104 and pPT79 and the other, encoded by pPT71 and cadB, is a protein containing 12 transmembrane segments linked by hydrophilic segments of variable length with the NH2- and COOH-termini located in the cytoplasm (Fig. 1). Polyamine transporters encoded by pPT104 (spermidine-preferential uptake system) and by pPT79 (putrescine-specific uptake system) are ABC transporters. The spermidine preferential uptake system consists of four proteins: PotA (an ATPase), PotB and PotC (channel-forming proteins), and PotD (a substrate-binding protein) (15). Similarly, the putrescine-uptake system also consists of four proteins: PotF (a substrate-binding protein), PotG (an ATPase), and PotH and PotI (channel-forming proteins) (16).

To determine whether all four proteins are necessary for spermidine uptake in the PotA/B/C/D system, the genes for each protein were individually disrupted by inserting the gene for kanamycin resistance in the Pot protein gene. Spermidine uptake was not observed in E. coli in which any one of the four proteins was disrupted. Transformation with a plasmid containing an intact gene corresponding to the disrupted one restored spermidine-uptake activity. Thus all four proteins (PotA, PotB, PotC, and PotD) are necessary for spermidine uptake. The calculated molecular masses of PotA, PotB, PotC, and PotD were 43, 31, 29, and 39 kDa, respectively (15). Based on hydrophobicity analysis, PotB and PotC contain six putative transmembrane segments linked by hydrophilic segments of variable length. PotA and PotD do not contain notable hydrophobic segments. When the amino acid sequence of PotA was compared with that of other proteins, a consensus nucleotide-binding sequence was found in PotA similar to that seen in the a and P subunits of E. coli ATPase (17), HisP and MalK proteins (18). Both HisP and MalK are membrane-associated ATPases of the histidine and maltose transport systems. We therefore speculated that PotA may be also membrane associated, and, indeed, it was shown that PotA exists mainly in the inner membrane fraction. PotA was associated with membranes through the interaction with

PotB and PotC (19). Because PotA was suggested to be involved in an energy-coupling step, like HisP and MalK (18), the ATP dependency of spermidine uptake was examined. ATP was found to be essential for spermidine uptake (26). The ATPase activity of PotA was studied using purified PotA and a PotABC complex on inside-out membrane vesicles. It was found that PotA can form a dimer by disulfide crosslinking, but that each PotA molecule functions independently (21). This is in accordance with previous results with HisP (22,23), although positive cooperativity for ATP during ATP hydrolysis was reported in HisP (24). When PotA was associated with the membrane proteins PotB and PotC, the Km value for ATP increased from 0.39 to 1.49 mM, and PotA became much more sensitive to inhibition by spermidine. The Ki value for spermidine was approx 10 |M at the Pot ABC complex, and spermidine uncompetitively inhibited PotA activity, suggesting that spermidine binds at a site on PotA different from the ATP-recognizing site. The results suggest that spermidine functions as a feedback inhibitor of spermidine uptake through inhibition of the ATPase activity of PotA. Amino acid residues involved in the ATPase activity were then identified (Fig. 2) and corresponded to Cys54, Val135, and Asp172. These results indicate that the amino acid residues necessary for ATP hydrolysis of PotA are located both within and between the two consensus amino acid sequences for nucleotide binding (GPSGC54GKT and LLLLD172E). Because the homology of amino acid sequences between PotA and HisP is relatively high, the important amino acid residues in PotA were tentatively placed on the HisP structure determined by X-ray crystal analysis (25). Although Cys54 and Asp172 are located at the ATP-binding domain, the position of Val135 is distant from the ATP-binding domain, suggesting that Val135 may be important for the structure of the active site of ATP hydrolysis. HisP (258 amino acid residues) is smaller than PotA (378 amino acid residues) and MalK (372 amino acid residues). It has been reported that the COOH terminus of MalK is critical for negative regulation of the mal operon (26), and that a mutant (E306K) of the COOH terminus of MalK affects its ATPase activity, suggesting a role for this region in the ATPase activity (27). Similarly, a mutant (E297K) of the COOH terminus of PotA and the COOH-terminal truncated mutants C1 (301 amino acid residues) and C2 (239 amino acid residues) affect its ATPase activity and the spermidine inhibition of ATPase activity (21). Thus there are two domains in PotA like MalK. The NH2-terminal domain (residues 1-250) contains the ATP-binding pocket formed in part by residues Cys26 (binding site of 8-azide-ATP), Phe27, Phe45, Cys54, Leu60, and Leu76, and the active center of the ATPase that includes Val135 and Asp172. The COOH-terminal domain (residues 251-378) of PotA contains a site that regulates ATPase activity and a site involved in the spermidine inhibition of ATPase activity.

PotD was purified to homogeneity, and the NH2-terminal sequence was determined by Edman degradation. The processing site of PotD by signal peptidase was between Ala23 and Asp24. Dissociation constants of spermidine and putrescine for purified PotD under the condition of 1 mM Mg2+ and 100 mM K+ at pH 7.5 were 3.2 and 100 |_iM, respectively. These values reflect the uptake system in intact cells. There was a single binding site for spermidine or putrescine on PotD, and spermidine uptake was shown to be PotD-dependent using right-side-out membrane vesicles. The PotD protein, as with

Fig. 2. Spermidine uptake and ATPase activities of various PotA mutants (A) and the position of mutations in the PotA mutants (B). (Reproduced with permission from ref. 21.)

other periplasmic binding proteins, consists of two domains with alternating P-a-P topology (28) with the polyamine-binding site located in a central cleft between these domains. Four acidic residues recognize the three positively charged nitrogen atoms of spermidine, and five aromatic side chains anchor the methylene backbone by van der

Waals interactions (Fig. 3). The overall fold of PotD is similar to that of other periplas-mic-binding proteins, in particular to the maltodextrin-binding protein from E. coli (29), even though the sequence identity is low. The crystal structure in the absence of spermidine is not known. However, a comparison of the PotD-spermidine structure with that of the maltodextrin-binding protein, determined in the presence and absence of its substrate, suggests that the binding of spermidine rearranges the relative orientation of the PotD domains to create a more compact structure. It was found that 13 amino acid residues were involved in binding of spermidine (Fig. 3). Among these residues, Glu171, Trp255, and Asp257 were the most important for binding of spermidine, Trp34, Tyr85, Asp168, Trp229, and Tyr293 had moderate contributions and the other five amino acids made a weak contribution to spermidine binding (30). The dissociation constants of spermidine for PotD mutated at Glu171, Trp255, and Asp257 increased greatly compared with the other mutants. Because these three residues interact with the diaminopropane moiety of spermidine, the results agree with the finding that PotD has a higher affinity for spermidine than for putrescine. Similarly, using PotD mutants, putrescine was found to bind at the position of the diaminobutane moiety of spermidine.

Properties of the putrescine binding protein (PotF) in the putrescine-specific uptake system was also clarified by the crystal structure together with studies of mutated PotF proteins (31). The structure of PotF is reminiscent of other periplasmic substrate binding proteins, with the highest structural similarity to that of PotD. Putrescine was tightly bound in a deep cleft between the two domains of PotF. The structure revealed the residues crucial for putrescine binding (Trp37, Ser85, Trp244, Asp247, and Asp278) and the importance of water molecules for putrescine recognition. Two residues in PotD, Thr35 and Glu36, the side chains of which make hydrogen bonds with the N1 nitrogen of spermidine, are replaced by Ser38 and Asp39 in PotF. In contrast to PotD, putrescine makes hydrogen bonds with the main chain carbonyl oxygen of these two residues. The side chain of Ser38 interacts with the N1 nitrogen of putrescine through the water molecule. Moreover, in PotF, the carboxyl oxygen atoms of Asp247 make both direct and water-mediated hydrogen bonds with the N1 atom of putrescine. Thus binding at the N1 site of putrescine in PotF is much stronger than that observed in PotD. This observation may explain the lower affinity of PotD than PotF for putrescine because the other interactions of the proteins with putrescine (i.e., the diaminobutane portion of spermidine) are very similar. In PotF, the position of the N1 atom of putrescine is strictly fixed, whereas in PotD, the N1 atom of spermidine is more flexible. To understand whether this difference may prevent binding of spermidine to PotF, we made a docking model of the spermidine molecule with a fixed position of its N1 amino group. There was no conformation that lacked steric hindrance with some amino acid residues in the PotF-binding sites. Thus we presume that the substrate selectivity of PotF is dominated by the unique hydrogen bond network with the N1 amino group, such that polyamines larger than putrescine can not fit into the PotF-binding cavity.

The crystal structures of PotD and PotF provide the first insight into the molecular mechanism by which proteins bind to and discriminate between different polyamines. The structural results, in combination with the mutational analyses, revealed that polyamine recognition could be achieved by the cooperation of multiple polar and

Polyamine Binds Dna


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