Spermidine-preferential Uptake System Research Articles

Effect of Spermidine Analogues on Cell Growth of Escherichia coli Polyamine Requiring Mutant MA261.

The effects of spermidine analogues [norspermidine (NSPD, 33), spermidine (SPD, 34), homospermidine (HSPD, 44) and aminopropylcadaverine (APCAD, 35)] on cell growth were studied using Escherichia coli polyamine-requiring mutant MA261. Cell growth was compared at 32°C, 37°C, and 42°C. All four analogues were taken up mainly by the PotABCD spermidine-preferential uptake system. The degree of stimulation of cell growth at 32°C and 37°C was NSPD ≥ SPD ≥ HSPD > APCAD, and SPD ≥ HSPD ≥ NSPD > APCAD, respectively. However, at 42°C, it was HSPD » SPD > NSPD > APCAD. One reason for this is HSPD was taken up effectively compared with other triamines. In addition, since natural polyamines (triamines and teteraamines) interact mainly with RNA, and the structure of RNA is more flexible at higher temperatures, HSPD probably stabilized RNA more tightly at 42°C. We have thus far found that 20 kinds of protein syntheses are stimulated by polyamines at the translational level. Among them, synthesis of OppA, RpoE and StpA was more strongly stimulated by HSPD at 42°C than at 37°C. Stabilization of the initiation region of oppA and rpoE mRNA was tighter by HSPD at 42°C than 37°C determined by circular dichroism (CD). The degree of polyamine stimulation of OppA, RpoE and StpA synthesis by NSPD, SPD and APCAD was smaller than that by HSPD at 42°C. Thus, the degree of stimulation of cell growth by spermidine analogues at the different temperatures is dependent on the stimulation of protein synthesis by some components of the polyamine modulon.

Synthesis and antimicrobial activity of chloramphenicol–polyamine conjugates

A series of chloramphenicol (CAM) amides with polyamines (PAs), suitable for structure–activity relationship studies, were synthesized either by direct attachment of the PA chain on the 2-aminopropane-1,3-diol backbone of CAM, previously oxidized selectively at its primary hydroxyl group, or from chloramphenicol base (CLB) through acylation with succinic or phthalic anhydride and finally coupling with a PA. Conjugates 4 and 5, in which the CLB moiety was attached on N4 and N1 positions, respectively, of the N8,N8-dibenzylated spermidine through the succinate linker, were the most potent antibacterial agents. Both conjugates were internalized into Escherichia coli cells by using the spermidine-preferential uptake system and caused decrease in protein and polyamine content of the cells. Noteworthy, conjugate 4 displayed comparable activity to CAM in MRSA or wild-type strains of Staphylococcus aureus and Escherichia coli, but superior activity in E. coli strains possessing ribosomal mutations or expressing the CAM acetyltransferase (cat) gene. Lead compounds, and in particular conjugate 4, have been therefore discovered during the course of the present work with clinical potential.

Crystallization and preliminary crystallographic studies of PotA, a membrane-associated ATPase of the spermidine-preferential uptake system inThermotoga maritima

A membrane-associated ATPase, PotA, is a component of the spermidine-preferential uptake system in prokaryotes that plays an important role in normal cell growth by regulating the cellular polyamine concentration. No three-dimensional structures of membrane-associated ATPases in polyamine-uptake systems have been determined to date. Here, the crystallization and preliminary X-ray diffraction analysis of PotA from Thermotoga maritima are reported. Diffraction data were collected and processed to 2.7 Å resolution from both native and selenomethionine-labelled crystals. Preliminary crystallographic analysis revealed that the crystals belonged to the hexagonal space group P3₁12 (or P3₂12), with unit-cell parameters a=b=88.9, c=221.2 Å, α=90, β=90, γ=120°, indicating that a dimer was present in the asymmetric unit.

Identification and Functions of Amino Acid Residues in PotB and PotC Involved in Spermidine Uptake Activity

Amino acid residues on PotB and PotC involved in spermidine uptake were identified by random and site-directed mutagenesis. It was found that Trp(8), Tyr(43), Trp(100), Leu(110), and Tyr(261) in PotB and Trp(46), Asp(108), Glu(169), Ser(196), Asp(198), and Asp(199) in PotC were strongly involved in spermidine uptake and that Tyr(160), Glu(172), and Leu(274) in PotB and Tyr(19), Tyr(88), Tyr(148), Glu(160), Leu(195), and Tyr(211) in PotC were moderately involved in spermidine uptake. Among 11 amino acid residues that were strongly involved in spermidine uptake, Trp(8) in PotB was important for insertion of PotB and PotC into membranes. Tyr(43), Trp(100), and Leu(110) in PotB and Trp(46), Asp(108), Ser(196), and Asp(198) in PotC were found to be involved in the interaction with PotD. Leu(110) and Tyr(261) in PotB and Asp(108), Asp(198), and Asp(199) in PotC were involved in the recognition of spermidine, and Trp(100) and Tyr(261) in PotB and Asp(108), Glu(169), and Asp(198) in PotC were involved in ATPase activity of PotA. Accordingly, Trp(100) in PotB was involved in both PotD recognition and ATPase activity, Leu(110) in PotB was involved in both PotD and spermidine recognition, and Tyr(261) in PotB was involved in both spermidine recognition and ATPase activity. Asp(108) and Asp(198) in PotC were involved in PotD and spermidine recognition as well as ATPase activity. These results suggest that spermidine passage from PotD to the cytoplasm is coupled to the ATPase activity of PotA through a structural change of PotA by its ATPase activity.

Sequence Analysis of Putative potB, potC, and potD Genes from Serratia rubidae

Amplification of putative potBCD genes from Serratia rubidae was conducted by PCR using a pair of primers FERC and RERC. A fragment with ~1800 bp size was ligated to pGEM-T Easy vector then cloned to competent Escherichia coli DH5α. The recombinant plasmid was sequenced using SP6, T7 and two internal primers, FPC and RPC. A sequence similarity search and analysis was performed with the BLASTN program. The sequence was found to have 83% similarity to potABCD genes from E. coli. Those genes encoded a spermidine-preferential uptake system that consists of four kinds of protein: PotA is a membrane-associated ATPase, PotB and PotC are transmembrane proteins that form channels, and PotD is a periplasmic substrate-binding protein. Alignment analysis showed that the isolated clone consisted of potB (partial), potC (full length) and potD (partial). The sequences for potBCD genes from S. rubidae are not available in the NCBI database. Furthermore, we have submitted this sequence, potBCD from S. rubidae , on GeneBank with Acc. number FJ447342.

The ATPase Activity and the Functional Domain of PotA, a Component of the Spermidine-preferential Uptake System inEscherichia coli

The ATPase activity of PotA, a component of the spermidine-preferential uptake system consisting of PotA, -B, -C, and -D, 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 cross-linking but that each PotA molecule functions independently. When PotA was associated with the membrane proteins PotB and PotC, the K(m) value for ATP increased and PotA became much more sensitive to inhibition by spermidine. It was also shown that spermidine uptake in cells was gradually inhibited in parallel with spermidine accumulation in cells. The results suggest that spermidine functions as a feedback inhibitor of spermidine transport. The function of PotA was analyzed using PotA mutants obtained by random mutagenesis. There are two domains in PotA. The NH2-terminal domain (residues 1-250) contains the ATP binding pocket formed in part by residues Cys26, Phe27, Phe45, Cys54, Leu60, and Leu76, the active center of ATPase that includes Val135 and Asp172, and amino acid residues necessary for the interaction with a second PotA subunit (Cys26) and with PotB (Cys54). 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.

Polyamine transport in bacteria and yeast

The polyamine content of cells is regulated by biosynthesis, degradation and transport. In Escherichia coli, the genes for three different polyamine transport systems have been cloned and characterized. Two uptake systems (putrescine-specific and spermidine-preferential) were ABC transporters, each consisting of a periplasmic substrate-binding protein, two transmembrane proteins and a membrane-associated ATPase. The crystal structures of the substrate-binding proteins (PotD and PotF) have been solved. They consist of two domains with an alternating β-α-β topology, similar to other periplasmic binding proteins. The polyamine-binding site is in a cleft between the two domains, as determined by crystallography and site-directed mutagenesis. Polyamines are mainly recognized by aspartic acid and glutamic acid residues, which interact with the NH2- (or NH-) groups, and by tryptophan and tyrosine residues that have hydrophobic interactions with the methylene groups of polyamines. The precursor of one of the substrate binding proteins, PotD, negatively regulates transcription of the operon for the spermidine-preferential uptake system, thus providing another level of regulation of cellular polyamines. The third transport system, catalysed by PotE, mediates both uptake and excretion of putrescine. Uptake of putrescine is dependent on membrane potential, whereas excretion involves an exchange reaction between putrescine and ornithine. In Saccharomyces cerevisiae, the gene for a polyamine transport protein (TPO1) was identified. The properties of this protein are similar to those of PotE, and TPO1 is located on the vacuolar membrane.

Polyamine transport in bacteria and yeast

The polyamine content of cells is regulated by biosynthesis, degradation and transport. In Escherichia coli, the genes for three different polyamine transport systems have been cloned and characterized. Two uptake systems (putrescine-specific and spermidine-preferential) were ABC transporters, each consisting of a periplasmic substrate-binding protein, two transmembrane proteins and a membrane-associated ATPase. The crystal structures of the substrate-binding proteins (PotD and PotF) have been solved. They consist of two domains with an alternating beta-alpha-beta topology, similar to other periplasmic binding proteins. The polyamine-binding site is in a cleft between the two domains, as determined by crystallography and site-directed mutagenesis. Polyamines are mainly recognized by aspartic acid and glutamic acid residues, which interact with the NH(2)- (or NH-) groups, and by tryptophan and tyrosine residues that have hydrophobic interactions with the methylene groups of polyamines. The precursor of one of the substrate binding proteins, PotD, negatively regulates transcription of the operon for the spermidine-preferential uptake system, thus providing another level of regulation of cellular polyamines. The third transport system, catalysed by PotE, mediates both uptake and excretion of putrescine. Uptake of putrescine is dependent on membrane potential, whereas excretion involves an exchange reaction between putrescine and ornithine. In Saccharomyces cerevisiae, the gene for a polyamine transport protein (TPO1) was identified. The properties of this protein are similar to those of PotE, and TPO1 is located on the vacuolar membrane.

Spermidine-preferential Uptake System in Escherichia coli: IDENTIFICATION OF AMINO ACIDS INVOLVED IN POLYAMINE BINDING IN PotD PROTEIN

Spermidine-binding sites on PotD protein, substrate-binding protein in periplasm, in the spermidine-preferential uptake system in Escherichia coli were studied by measuring polyamine transport activities of right-side-out membrane vesicles with mutated PotD proteins prepared by site-directed mutagenesis of the potD gene and by measuring polyamine binding activities of these mutated PotD proteins. Polyamine transport activities of the mutated PotD proteins paralleled their polyamine binding activities. It was found that Trp-34, Thr-35, Glu-36, Tyr-37, Ser-83, Tyr-85, Asp-168, Glu-171, Trp-229, Trp-255, Asp-257, Tyr-293, and Gln-327 of PotD protein were involved in the binding to spermidine. When spermidine uptake activities were measured in intact cells expressing the mutated PotD proteins, it was found that Glu-171, Trp-255, and Asp-257 were more strongly involved in the binding of spermidine to PotD protein than the other amino acids listed above. The dissociation constants of spermidine for the mutated PotD proteins at Glu-171, Trp-255, and Asp-257 increased greatly in comparison with those for the other mutated PotD proteins. Since these three amino acids clearly interact with the diaminopropane moiety of spermidine, the results are in accordance with the finding that PotD protein has a higher affinity for spermidine than for putrescine. Putrescine was found to bind at the position of the diaminobutane moiety of spermidine.

Polyamine transport inEscherichia coli

The polyamine content in cells is regulated by both polyamine biosynthesis and its transport. We recently obtained and characterized three clones of polyamine transport genes (pPT104, pPT79 and pPT71) inEscherichia coli. The system encoded by pPT104 was the spermidine-preferential uptake system and that encoded by pPT79 the putrescine-specific uptake system. Furthermore, these two systems were periplasmic transport systems consisting of four kinds of proteins: pPT104 clone encoded potA, -B,-C, and -D proteins and pPT79 clone encoded potF, -G, -H, and -I proteins, judging from the deduced amino acid sequences of the nucleotide sequences of these clones. PotD and -F proteins were periplasmic substrate binding proteins and potA and -G proteins membrane associated proteins having the nucleotide binding site. PotB and -C proteins, and potH and -I proteins were transmembrane proteins probably forming channels for spermidine and putrescine, respectively. Their amino acid sequences in the corresponding proteins were similar to each other. The functions of potA and -D proteins in the spermidine-preferential uptake system encoded by pPT104 clone were studied in detail through a combined biochemical and genetic approach. In contrast, the putrescine transport system encoded by pPT71 consisted of one membrane protein (potE protein) haveing twelve transmembrane segments, and was active in both the uptake and excretion of putrescine. The uptake was dependent on membrane potential, and the excretion was due to the exchange reaction between putrescine and ornithine.

Polyamine transport in Escherichia coli and eukaryotic cells

The polyamine content in cells is regulated by both polyamine biosynthesis and its transport. We recently obtained and characterized three clones of polyamine transport genes (pPT104, pPT79 and pPT71) in Escherichia coli. The system encoded by pPT104 was the spermidine-preferential uptake system and that encoded by pPT79 the putrescine-specific uptake system. Furthermore, these two systems were ABC (ATP binding cassette) transporters consisting of four kinds of proteins: pPT104 clone encoded PotA, -B, -C, and -D proteins and pPT79 clone encoded PotF, -G, -H, and I proteins. PotD and -F proteins were periplasmic substrate binding proteins and PotA and -G proteins membrane associated proteins having the nucleotide binding site. PotB and -C proteins, and PotH and -I proteins were transmembrane proteins probably forming channels for spermidine and putrescine, respectively. Their amino acid sequences in the corresponding proteins were similar to each other. The functions of PotA and -D proteins in the spermidine-preferential uptake system encoded by pPT104 clone were studied in detail through a combined biochemical and genetic approach. In contrast, the putrescine transport system encoded by pPT71 consisted of one membrane protein (PotE protein) having twelve transmembrane segments, and was active in both the uptake and excretion of putrescine. The uptake was dependent on the membrane potential, and the excretion was due to the exchange reaction between putrescine and ornithine. In mouse mammary carcinoma FM3A cells, it was shown that the antizyme, which negatively regulates the amount of ornithine decarboxylase, also negatively regulates the activity of polyamine transport.

Spermidine-preferential Uptake System in Escherichia coli: ATP HYDROLYSIS BY PotA PROTEIN AND ITS ASSOCIATION WITH MEMBRANES

PotA protein, one of the components of the spermidine-preferential uptake system in Escherichia coli, was purified to homogeneity, and some of its properties were examined. PotA protein showed Mg(2+)-and SH-dependent ATPase activity. The specific activity was approximately 400 nmol/min/mg of protein and the Km value for ATP was 385 microM. The nature of the ATP binding site was explored by identification of the amino acid residue photoaffinity-labeled with 8-azido-ATP. It was found that 8-azido-ATP was attached to cysteine 26. In the spermidine transport-deficient mutant E. coli NH1596, valine 135 of PotA protein, which is located between two consensus amino acid sequences for nucleotide binding (50-57 and 168-173), was replaced by methionine (Kashiwagi, K., Miyamoto, S., Nukui, E., Kobayashi, H., and Igarashi, K. (1993) J. Biol. Chem. 268, 19358-19363). This mutated PotA protein could be labeled with 8-azido-ATP, but showed very low ATPase activity. To identify which cysteine is involved in the function of potA protein, cysteines 26, 54, and 276 were replaced by alanine, threonine, and alanine, respectively. Among the three mutated PotA proteins, the mutated PotA protein C54T only lost both ATPase and spermidine uptake activities. The results taken together indicate that the adenine portion of ATP interacts with a domain close to the NH2-terminal end of PotA protein, and active centers of ATP hydrolysis are located both within and between the two consensus amino acid sequences for nucleotide binding. Association of PotA protein with membranes was strengthened by the existence of channel forming PotB and PotC proteins. ATPase of PotA protein was inhibited by spermidine, suggesting that uptake inhibition by spermidine may function during this process.

Properties and structure of spermidine acetyltransferase in Escherichia coli.

Spermidine acetyltransferase (SAT) from Escherichia coli was purified about 40,000-fold. The molecular mass of native SAT was 95 kDa, and it consisted of four identical subunits. The products formed from the reaction of acetyl-CoA with spermidine by SAT were N1- and N8-acetylspermidine. The Km values for acetyl-CoA, spermidine, and spermine were 2 microM, 1.29 mM, and 220 microM, respectively. The enzymatic activity increased by 2.5-3.5-fold under the condition of poor nutrition but not in response to cold shock or high pH. By using synthetic oliogonucleotides deduced from amino acid sequences of the peptides in SAT, a polymerase chain reaction product with a length of 250 nucleotides was obtained. Using this polymerase chain reaction product, the gene encoding SAT (speG) was cloned and mapped at 35.6 min in the E. coli chromosome. E. coli cells transformed with the cloned speG gene increased SAT activity by 8-40-fold. The gene encoded a 186-amino acid protein, but SAT consisted of 185 amino acids because the initiator methionine was liberated from the protein. Thus, the predicted molecular mass was 21,756 Da. Significant similarity to aminoglycoside acetyltransferase and peptide N-acetyltransferase was observed in the amino acid sequence 87-141, and some similarity with spermidine-preferential binding protein (potD protein) in the spermidine-preferential uptake system was observed in the amino acid sequence 122-141. The results suggest that the active center of SAT may be located in the COOH-terminal portion.

Functions of potA and potD proteins in spermidine-preferential uptake system in Escherichia coli.

Functions of potA and -D proteins in the spermidine-preferential uptake system, which consists of potA, -B, -C, and -D proteins, were studied. Spermidine uptake activity was lost when the gene for potA or potD protein was disrupted, and transformation of the cells with potA or potD gene recovered the uptake activity. PotD protein was found to bind spermidine with a 3.2 microM dissociation constant. Spermidine uptake by membrane vesicles prepared from Escherichia coli DR112 containing the genes for potA, -B, and -C proteins was strongly dependent on the addition of potD protein, and its optimal concentration was 5 microM when 10 microM spermidine was used as substrate. The ATP dependence of spermidine uptake was examined with the atp mutant of E. coli. The uptake was completely dependent on ATP. When the membrane potential was extinguished by carbonyl cyanide m-chlorophenylhydrazone, the uptake activity was decreased by 60% even if ATP existed. This suggests that the membrane potential is also involved in the spermidine uptake. ATP was found to bind to potA protein. In the spermidine transport-deficient mutant E. coli NH1596, valine 135 of potA protein, which is located between two consensus amino acid sequences for nucleotide binding, was replaced by methionine. Although the amount of mutated potA protein expressed in E. coli cells was the same as that of normal potA protein and the mutated protein was membrane-associated, no significant spermidine uptake was observed. The results taken together indicate that potA and -D proteins are absolutely necessary for spermidine uptake in conjunction with the two channel forming proteins (potB and -C).