Identification of a Novel Aminopropyltransferase Involved in the Synthesis of Branched-Chain Polyamines in Hyperthermophiles
Identification of a Novel Aminopropyltransferase Involved in the Synthesis of Branched-Chain Polyamines in Hyperthermophiles
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DOI：
10.1128/jb.01515-14
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发表时间：
2014-05-01
影响因子：
3.2
通讯作者：
Fujiwara, Shinsuke
中科院分区：
文献类型：
--
作者：
Okada, Kazuma;Hidese, Ryota;Fujiwara, Shinsuke
长链和/或支链多胺是在嗜热菌中发现的独特的聚阳离子。N-4-氨丙基精胺被认为是Kodakarensis中的主要多胺。为了确定是否还存在季支化五胺，N-4-双（氨丙基）亚精胺，N-4-氨丙基精胺的异构体，酸提取的细胞质多胺通过高压液相色谱法，气相色谱法（HPLC）和气相色谱-质谱法进行了分析。N-4-双（氨丙基）亚精胺是该物种丰富的细胞质多胺。为了鉴定催化N-4-双（氨丙基）亚精胺合成的酶，从细胞质中浓缩活性级分，并在通过MASCOT数据库分析后，用电喷雾电离仪通过线性离子阱-飞行时间质谱法进行分析。TK0545、TK0548、TK0967和TK1691被鉴定为候选酶，并且相应的基因被单独克隆并在大肠杆菌中表达。纯化重组形式，并测量其N-4-双（氨丙基）亚精胺合成活性。在四个候选菌株中，发现TK 1691（BpsA）可以从亚精胺经N-4-氨丙基亚精胺合成N-4-双（氨丙基）亚精胺。与野生型相比，bpsA破坏的菌株DBP1在85 ℃下生长，滞后期稍长，但在93 ℃下无法生长。HPLC分析表明，在85 ℃下生长的DBP 1菌株中不存在N-4-氨丙基亚精胺和N-4-双（氨丙基）亚精胺，这表明由BpsA合成的支链多胺对于93 ℃下的细胞生长是重要的。与来自各种微生物的直系同源物的序列比较表明，BpsA不同于其他已知的产生亚精胺和精胺的氨丙基转移酶。BpsA直系同源物仅在嗜热菌中发现，在古细菌和细菌中，但在嗜温菌中不存在。这些发现表明BpsA是合成支链多胺所必需的一种新型氨丙基转移酶，使嗜热菌能够在高温环境中生长。
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 N4-bis(aminopropyl) spermidine from spermidine via N-4-aminopropylspermidine. Compared to the wild type, the bpsA-disrupted strain DBP1 grew at 85 degrees C with a slightly longer lag phase but was unable to grow at 93 degrees C. HPLC analysis showed that both N-4-aminopropylspermidine and N-4-bis(aminopropyl) spermidine were absent from the DBP1 strain grown at 85 degrees C, demonstrating that the branched-chain polyamine synthesized by BpsA is important for cell growth at 93 degrees 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.