The pK values of thermospermine, a novel asymmetric tetraamine (4,8-diazadodecane-l,12-diamine), have been determined for the first time by the conventional titration method with subsequent data analyses. It was found to be the most basic polyamine among naturally occurring tetraamines. The I5N N M R titration method was applied to determine its protonation site. Natural-abundance I5N magnetic resonance spectra of the polyamine have been recorded as a function of pH from its free to the protonated form. Its apparent pK values, as well as the intrinsic chemical shifts for each stage of protonation, have been calculated from the N M R titration curve by using a nonlinear least-squares method. The four protonation sites of thermospermine have been successfully determined to be Nt2, N', N-8, and N-4 in chat order from the free to the completely protonated form. In a similar manner the protonation sites of spermidine and two symmetric tetraamines, thermine and spermine, have been established. I3C N M R experiments for the four polyamines were almost consistent with those of I5N NMR. Owing to the much higher concentration of the polyamines furnished for N M R titration experiments, the pK values obtained by NMR were larger than those derived by the conventional titration methods.A considerable number of observations show a correlation of enhanced synthesis of polyamines with rapid growth or cell proliferation [1,2]. From the point of view of the physiological role of polyamines in cells, polyamines were implicated as regulators of cell proliferative activity [3-S]. Thus it is tempting to speculate that association as well as dissociation of polyamines with biological macromolecules may be controlled dynamically. In fact, it has been shown that polyamines can bind with nucleic acids [S -81 and that they exert a remarkable stabilization of a teritary structure of the nucleic acids [9 -121. This led us to determine the protonation sites in polyamines and the corresponding pK values, and to examine their effect on the interaction with biological macromolecules.The pK values of several naturally occurring and synthetic polyamines have been determined by means of potentiometric titration methods [I 3 -IS]. However, the pK values of thermospermine, i.e. 4,s-diazadodecane-l , 12-diamine have remained undetermined. This polyamine is a new asymmetric tetraamine which was found in an extremely thermophilic microorganism, Thermus tl~rrmnphilus, and its structure has been determined [16]. Thus we synthesized it and measured the p K values by potentiometric titration.Determination of the protonation sites in polyamines has been attempted by calorimetric measurements [I 7,181 and the calculation of protonation enthalpy by applying quantum chemical methods [I 91. These attempts were almost successfull, but they are indirect methods. NMR titration, on the other hand, provides pK values and information about the protonation pattern, since chemical shifts of the nuclei at the basic sites are often markedly influenced by the ionization o...

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...

Triamines such as norspermidine, spermidine, and homospermidine and tetraamines such as norspermine, spermine, thermospermine, and aminopropylhomospermidine were found to be distributed ubiquitously in the eight extremely thermophilic (growing at 70 degrees C) Thermus species tested. Three linear pentaamine (caldopentamine, homocaldopentamine, and thermopentamine), two linear hexaamines (caldohexamine and homocaldohexamine), two tertiary branched tetraamines (N4-aminopropylnorspermidine and N4-aminopropyl-spermidine), and quaternary branched pentaamines such as N4-bis(aminopropyl)norspermidine and N4-bis(aminopropyl)spermidine were detected in T. thermophilus HB8, T. filiformis Wai33 A1, T. flavus AT-62, and T. caldophilus GK24. The linear hexaamines and branched polyamines were absent in T. aquaticus YT-1, T. sp. X-1, T. sp. T2, and T. sp. T351, in which linear pentaamines were minor components. Moderately thermophilic Thermus ruber and Thermus sp. K-2 contained putrescine, spermidine, norspermidine, homospermidine, spermine, norspermine, thermospermine, and aminopropylhomospermidine. No pentaamines, hexaamines, or branched polyamines were found in these two moderately thermophilic Thermus species. On the other hand, moderately thermophilic, acidophilic Acidothermus cellulolyticus was devoid of all the polyamines.

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