Identification of a Novel Aminopropyltransferase Involved in the Synthesis of Branched-Chain Polyamines in Hyperthermophiles

Abstract

f Longer-and/or branched-chain polyamines are unique polycations found in thermophiles. N 4 -aminopropylspermine is considered a major polyamine in Thermococcus kodakarensis. To determine whether a quaternary branched penta-amine, N 4 -bis(aminopropyl)spermidine, an isomer of N 4 -aminopropylspermine, was also present, acid-extracted cytoplasmic polyamines were analyzed by high-pressure liquid chromatography, gas chromatography (HPLC), and gas chromatography-mass spectrometry. N 4 -bis(aminopropyl)spermidine was an abundant cytoplasmic polyamine in this species. To identify the enzyme that catalyzes N 4 -bis(aminopropyl)spermidine synthesis, the active fraction was concentrated from the cytoplasm and analyzed by linear ion trap-time of flight mass spectrometry with an electrospray ionization instrument after analysis by the MASCOT database. TK0545, TK0548, TK0967, and TK1691 were identified as candidate enzymes, and the corresponding genes were individually cloned and expressed in Escherichia coli. Recombinant forms were purified, and their N 4 -bis(aminopropyl)spermidine synthesis activity was measured. Of the four candidates, TK1691 (BpsA) was found to synthesize N 4 -bis(aminopropyl)spermidine from spermidine via N 4 -aminopropylspermidine. Compared to the wild type, the bpsA-disrupted strain DBP1 grew at 85°C with a slightly longer lag phase but was unable to grow at 93°C. HPLC analysis showed that both N 4 -aminopropylspermidine and N 4 -bis(aminopropyl)spermidine were absent from the DBP1 strain grown at 85°C, demonstrating that the branched-chain polyamine synthesized by BpsA is important for cell growth at 93°C. Sequence comparison to orthologs from various microorganisms indicated that BpsA differed from other known aminopropyltransferases that produce spermidine and spermine. BpsA orthologs were found only in thermophiles, both in archaea and bacteria, but were absent from mesophiles. These findings indicate that BpsA is a novel aminopropyltransferase essential for the synthesis of branched-chain polyamines, enabling thermophiles to grow in high-temperature environments. P olyamines are small, positively charged aliphatic molecules containing more than two amine residues present in almost all living organisms. Putrescine [4], spermidine [34], and spermine [343] are polyamines commonly observed in the cells of various living organisms, from viruses to humans (1-4). Polyamines are important in cell proliferation and cell differentiation (5, 6), as well as contributing to adaptation to various stresses (7). Interestingly, in addition to common polyamines, thermophiles contain two types of unusual polyamines as major polyamines. One type consists of long linear polyamines such as caldopentamine [3333] NH, N,. Because the relative amounts of long/branched-chain polyamines in cells of (hyper)thermophiles were found to increase as growth temperatures increased, these unique polyamines are regarded as supporting the growth of thermophilic microorganisms under high-temperature conditions (18)(19)(20). An in...

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Active site geometry of a novel aminopropyltransferase for biosynthesis of hyperthermophile‐specific branched‐chain polyamine

“…Recently, we identified a novel aminopropyltransferase, belonging to a new family of these enzymes, responsible for the biosynthesis of branched-chain polyamines in extracts of the hyperthermophilic archaeon Thermococcus kodakarensis grown at 85-93°C [15]. This novel enzyme, termed BpsA, appears to catalyze the sequential aminopropyl transfer reaction from decarboxylated S-adenosylmethionine (dcSAM) to the initial substrate spermidine, resulting in the production of N 4 -bis(aminopropyl)spermidine via an N 4 -aminopropylspermidine intermediate (Fig.…”

“…This novel enzyme, termed BpsA, appears to catalyze the sequential aminopropyl transfer reaction from decarboxylated S-adenosylmethionine (dcSAM) to the initial substrate spermidine, resulting in the production of N 4 -bis(aminopropyl)spermidine via an N 4 -aminopropylspermidine intermediate (Fig. The bpsA knockout strain was unable to grow at 93°C, indicating that branched-chain polyamine plays an essential role in survival at high temperatures [15]. The bpsA knockout strain was unable to grow at 93°C, indicating that branched-chain polyamine plays an essential role in survival at high temperatures [15].…”

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