Extreme thermophiles produce two types of unusual polyamine: long linear polyamines such as caldopentamine and caldohexamine, and branched polyamines such as quaternary ammonium compounds [e.g. tetrakis(3-aminopropyl)ammonium]. To clarify the physiological roles of long linear and branched polyamines in thermophiles, we synthesized them chemically and tested their effects on the stability of ds (double-stranded) and ss (single-stranded) DNAs and tRNA in response to thermal denaturation, as measured by differential scanning calorimetry. Linear polyamines stabilized dsDNA in proportion to the number of amino nitrogen atoms within their molecular structure. We used the empirical results to derive formulae that estimate the melting temperature of dsDNA in the presence of polyamines of a particular molecular composition. ssDNA and tRNA were stabilized more effectively by tetrakis(3-aminopropyl)ammonium than any of the other polyamines tested. We propose that long linear polyamines are effective to stabilize DNA, and tetrakis(3-aminopropyl)ammonium plays important roles in stabilizing RNAs in thermophile cells.
In the extreme thermophile Thermus thermophilus, a disruption mutant of a gene homologous to speB (coding for agmatinase ؍ agmatine ureohydrolase) accumulated N 1 -aminopropylagmatine (N 8 -amidino-1,8-diamino-4-azaoctane, N 8 -amidinospermidine), a new compound, whereas all other polyamines produced by the wild-type strain were absent from the cells. Double disruption of speB and speE (polyamine aminopropyltransferase) resulted in the disappearance of N 1 -aminopropylagmatine and the accumulation of agmatine. These results suggested the following. 1) N 1 -Aminopropylagmatine is produced from agmatine by the action of an enzyme coded by speE. 2) N 1 -Aminopropylagmatine is a metabolic intermediate in the biosynthesis of unique polyamines found in the thermophile. 3) N 1 -Aminopropylagmatine is a substrate of the SpeB homolog. They further suggest a new biosynthetic pathway in T. thermophilus, by which polyamines are formed from agmatine via N 1 -aminopropylagmatine. To confirm our speculation, we purified the expression product of the speB homolog and confirmed that the enzyme hydrolyzes N 1 -aminopropylagmatine to spermidine but does not act on agmatine.Polyamines play important roles in cell proliferation and cell differentiation. Common polyamines such as putrescine, spermidine, and spermine are distributed ubiquitously in cells and tissues at relatively high concentrations (1, 2).Thermus thermophilus, of which the genome project was completed using two strains, HB8 and HB27 (Structural-Biological Whole Cell Project at www.srg.harima.riken.go.jp/ thermus/j_index.htm and see Ref. 3, respectively), produces a variety of polyamines including unusually long polyamines and branched ones (4) (see Fig. 9C). These long and branched polyamines have a marked effect of protecting and stabilizing nucleic acids (5, 6) and of activating cell-free polypeptide synthesis at high temperature (7-9).In many organisms, such as bacteria, yeast, animals, and plants, the first step of polyamine biosynthesis is production of putrescine by decarboxylation of L-ornithine (see Fig. 9A). An additional or alternative pathway of putrescine biosynthesis that is often seen in plants and sometimes in bacteria is decarboxylation of L-arginine followed by hydrolysis of agmatine. Agmatine ureohydrolase or agmatinase, coded by the speB gene, catalyzes this second reaction. The next step is production of spermidine and spermine by the addition of an aminopropyl group to putrescine and spermidine, respectively. This reaction is catalyzed by spermidine or spermine synthase (putrescine/spermidine aminopropyltransferase) coded by the speE gene (1).To investigate the polyamine biosynthetic pathway in T. thermophilus, we constructed a disruption strain of the speB gene homolog of T. thermophilus. Disruption of the speB gene homolog resulted in drastic reduction of triamines, longer and branched polyamines without accumulation of agmatine, and in accumulation of an unknown compound. Double disruption of speB and speE gene homologs resulted in disappear...
The binding of spermine and ifenprodil to the amino terminal regulatory (R) domain of the N‐methyl‐D‐aspartate receptor was studied using purified regulatory domains of the NR1, NR2A and NR2B subunits, termed NR1‐R, NR2A‐R and NR2B‐R. The R domains were over‐expressed in Escherichia coli and purified to near homogeneity. The Kd values for binding of [14C]spermine to NR1‐R, NR2A‐R and NR2B‐R were 19, 140, and 33 μM, respectively. [3H]Ifenprodil bound to NR1‐R (Kd, 0.18 μM) and NR2B‐R (Kd, 0.21 μM), but not to NR2A‐R at the concentrations tested (0.1–0.8 μM). These Kd values were confirmed by circular dichroism measurements. The Kd values reflected their effective concentrations at intact NR1/NR2A and NR1/NR2B receptors. The results suggest that effects of spermine and ifenprodil on NMDA receptors occur through binding to the regulatory domains of the NR1, NR2A and NR2B subunits. The binding capacity of spermine or ifenprodil to a mixture of NR1‐R and NR2A‐R or NR1‐R and NR2B‐R was additive with that of each individual R domain. Binding of spermine to NR1‐R and NR2B‐R was not inhibited by ifenprodil and vice versa, indicating that the binding sites for spermine and ifenprodil on NR1‐R and NR2B‐R are distinct.
The open reading frames (ORFs) TK0240, TK0474, and TK0882, annotated as agmatine ureohydrolase genes, were disrupted. Only the TK0882 gene disruptant showed a growth defect at 85°C and 93°C, and the growth was partially retrieved by the addition of spermidine. In the TK0882 gene disruptant, agmatine and N 1 -aminopropylagmatine accumulated in the cytoplasm. Recombinant TK0882 was purified to homogeneity, and its ureohydrolase characteristics were examined. It possessed a 43-fold-higher k cat /K m value for N 1 -aminopropylagmatine than for agmatine, suggesting that TK0882 functions mainly as N 1 -aminopropylagmatine ureohydrolase to produce spermidine. TK0147, annotated as spermidine/spermine synthase, was also studied. The TK0147 gene disruptant showed a remarkable growth defect at 85°C and 93°C. Moreover, large amounts of agmatine but smaller amounts of putrescine accumulated in the disruptant. Purified recombinant TK0147 possessed a 78-fold-higher k cat /K m value for agmatine than for putrescine, suggesting that TK0147 functions primarily as an aminopropyl transferase to produce N 1 -aminopropylagmatine. In T. kodakarensis, spermidine is produced mainly from agmatine via N 1 -aminopropylagmatine. Furthermore, spermine and N 4 -aminopropylspermine were detected in the TK0147 disruptant, indicating that TK0147 does not function to produce spermine and long-chain polyamines.
Polyamines play important roles in cell growth mainly through their interaction with RNA. We have previously reported that polyamines stimulate the synthesis of oligopeptide-binding protein OppA in Escherichia coli and the formation of Ile-tRNA in rat liver (Igarashi, K., and Kashiwagi, K. (2000) Biochem. Biophys. Res. Commun. 271, 559 -564). These effects involve an interaction of polyamines with the bulged-out region of double-stranded RNA in the initiation region of OppA mRNA and in the acceptor stem of rat liver tRNA Ile . In this study, the effects of polyamines on E. coli OppA synthesis and rat liver Ile-tRNA formation were compared using OppA mRNA and tRNA Ile with or without the bulged-out region of doublestranded RNA. The results indicate that the bulged-out region is involved in polyamine stimulation of OppA synthesis and IletRNA formation. A selective structural change by spermidine in the bulged-out region of double-stranded RNA was confirmed by circular dichroism.Polyamines (putrescine, spermidine, and spermine) are essential for normal cell growth due to effects mainly at the level of translation (1-3). It is known that polyamines bind preferentially to double-stranded RNA rather than single-stranded RNA and double-stranded DNA (4). Indeed, polyamines were found mostly in polyamine-RNA complexes when measured in rat liver, bovine lymphocytes, and Escherichia coli (5, 6). We reported previously that polyamines have not only a sparing effect on the Mg 2ϩ requirement of polyphenylalanine and globin synthesis but also a stimulatory effect that cannot be fulfilled by any amount of Mg 2ϩ in the absence of polyamines (7,8). It has also been reported that polyamines enhance, at the level of translation, the synthesis of several kinds of proteins that are important for cell growth in E. coli (3, 9 -11). We propose that a group of genes whose expression is enhanced by polyamines at the level of translation be referred to as the "polyamine modulon" (3).There are three different mechanisms underlying polyamine stimulation of the synthesis of various members of the polyamine modulon. First, polyamine stimulation of protein synthesis can occur when a Shine-Dalgarno sequence in the mRNA is obscure or is distant from the AUG initiation codon. In this case, polyamines cause structural changes in a region of the Shine-Dalgarno sequence and the AUG initiation codon of the mRNA, facilitating formation of the initiation complex, and examples include OppA, a periplasmic substrate-binding protein of the oligopeptide uptake system; FecI factor ( 18 ), for transcription of the iron transport operon; Fis, a global regulator of transcription of some growth-related genes, including those for rRNA and some tRNAs; RpoN factor ( 54 ), for transcription of genes for nitrogen metabolism; and H-NS, a transcription factor of many kinds of mRNAs, including ribosomal protein mRNAs and flagellar protein mRNAs. By a second mechanism, polyamines enhance the inefficient initiation codon UUG (or GUG)-dependent fMet-tRNA binding to Cya (or...
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