New regulation mechanism of polyamine biosynthesis mediated by ribosomal Protein, L10 as an antizyme

反刍硒单胞菌是一种严格的厌氧革兰氏阴性菌，它既没有游离形式的脂蛋白，也没有结合形式的脂蛋白，而在一般革兰氏阴性菌中，脂蛋白在维持细胞表面结构完整性方面起着重要作用。相反，它含有尸体碱，它与肽聚糖共价连接，作为细胞分裂的关键成分。尸胺是由l -赖氨酸通过赖氨酸脱羧酶（LDC）合成的，并作为正常细胞生长的必需成分转移到肽聚糖中。此外，南南团最不发达国家有两个独特的特点；(1)对l -赖氨酸和l -鸟氨酸均有脱羧酶活性。(2)生物化学前景与真核鸟氨酸相似。本研究发现：(1)s . rtm inanluon LDC在固定化早期被ATP依赖性丝氨酸蛋白酶显著降解。这种降解蛋白水解需要一个降解事实…More or，命名为P22。P22是由腐胺在S. raminanum细胞中积累引起的，它与LDC紧密结合，K_D为8.5X10^<-11> M. P22似乎与一种antizynte （AZ）具有相似的生化和生物物理特性，这仅在哺乳动物细胞中报道过。P22被鉴定为S. rimliuunlimn核糖体蛋白LI0。L10与LDC结合形成LDC-L10复合物可能引起LDC亚基的构象变化，使其受到atp依赖性丝氨酸蛋白酶的攻击，LDC将被分解成小肽。我们还确定了L-10分子中A区（K^<101>NKLD^<105>）和B区（G^<106>VIRNAVYVLD^<170>）与LDC结合所必需的两个区域。(2)我们发现了atp依赖的丝氨酸蛋白酶(s)，它参与了s . ruminantiian LDC的降解。反刍s.r inantium的atp依赖性蛋白酶染色体上有三个基因，我们重点研究了其中一个属于C IpXP蛋白酶的基因，因为它需要一个适配蛋白。CIpXP蛋白酶由CIpX atp酶和CIpP肽酶组成。在S. rminantitan中，L10可能作为一种适配器蛋白，将LDC携带到CIpXP蛋白酶。我们将S. ruminantiuni的C1pP基因克隆到pET15b中，在大肠杆菌BL21 （DE3）中表达并纯化。(3)与肽聚糖共价连接的尸胺是肽聚糖与主要外膜蛋白Mep45的s层同源（SLH）结构域相互作用所必需的。在这里，我们使用一系列一般结构为NH_3^+(CH_2)_nNH_3^+, n=3-6)的二胺，我们发现尸胺（n=5）特异性地作为肽聚糖的最有效成分，以获得细胞对外部损伤剂的高抗性，并且是Mep45的SLH结构域与肽聚糖之间有效相互作用所必需的，从而促进外膜正确锚定肽聚糖。少

Selenomonas ruminantium, which is a strictly anaerobic, Gram-negative bacterium has neither free nor bound forms of lipoprotein which plays an important role of the maintenance of the structural integrity of the cell surface in general Gram-negative bacteria. Instead, it has cadaverine, which links covalently to the pepticloglycan as a pivotal constituent for the cell devision. In S. rmuinantimn cadaverine is synthesized constitutively from L-lysine by lysine decarboxylase (LDC) and transferred to the peptidoglycan as an essential constituent for normal cell growth. Furthermore, S. rwninantuan LDC has two unique characteristics ; (1) it has decarboxylase activity towards both L-lysine and L-ornithine. (2) biochemical future is similar to that of eukaryotic ornithine. Here, we found the following evidences, i.e.(1) S. rtm inanluon LDC was dramatically degraded at the early stationary phase by ATP -dependent serine protease(s). This degradation proteolysis was required a degradation fact … More or, which was named as P22. P22 was induced by the accumulation of putrescine in S. raminanlium cells, and it bound tightly to LDC with a K_D) of 8.5X10^<-11> M. P22 seems to have similar biochemical and biophysical characteristics to those of an antizynte (AZ), which have been reported only in mammalian cells. P22 was identified as a ribosomal protein LI0 of S. rimliuunlimn. The binding of L10 to LDC for forming LDC-L10 complex may cause a conformational change(s) of LDC subunit, which allow it to be attacked by ATP-dependent serine protease(s), and the LDC would be broken down to small peptides. We also identified the two regions, A region (K^<101>NKLD^<105>) and B region (G^<106>VIRNAVYVLD^<170>) in L-10 molecule essential for its binding to LDC.(2) We identified the ATP-dependent serine protease(s), which is involved in the degradation of S. ruminantiian LDC. There were three genes for ATP-dependent protease chromosomal in S. ruminantium and we focused on one of them, which belongs to C IpXP protease(s) since they require an adapter protein. CIpXP protease consists of the CIpX ATPase and the CIpP peptidase. In S. rmninantitan, L10 may work as an adapter protein and carry the LDC to CIpXP protease. We cloned C1pP gene from S. ruminantiuni into pET15b and expressed it in E, coli BL21 (DE3) and purified.(3) Cadaverine covalently linked to the peptidoglycan is required for the interaction between the peptidoglycan and the S-layer homologous (SLH) domain of the major outer membrane protein, Mep45. Here, using a series of diamines with a general structure of NH_3^+(CH_2)_nNH_3^+, n=3-6), we found that cadaverine (n=5) specifically serves as the most efficient constituent of the peptidoglycan to acquire the high resistance of the cell to the external damage agents, and is required for the effective interaction between the SLH domain of Mep45 and the peptidoglycan facilitating the correct anchoring of the outer membrane to the peptidoglycan. Less