Heme-based sensor kinases in Methanosarcina acetivorans as a paradigm for archaeal signal transduction

产甲烷古菌Methanosarcina acetivorans的甲基硫甲基转移酶相关传感器MsmS（MA 4561）是一个多结构域传感器蛋白，由PAS和GAF结构域交替连接到C-末端组氨酸激酶样H_ATP酶输出结构域组成。MsmS在其第二GAF结构域中含有共价结合的血红素辅因子。这种血红素具有氧化还原活性，并且能够连接几个外部配体，包括一氧化碳和二甲基硫。血红素辅因子氧化后，H_ATP酶结构域的自磷酸化活性被诱导，表明MsmS是一种传感器激酶。借助msmS的染色体敲除突变体，我们可以进一步证明MsmS参与编码类咕啉/甲基转移酶同工酶的三个基因之一的交叉调节。这些基因的转录被证明是激活的上游编码的调节器的Msr家族。这些监管机构之一是编码下游的msmS，它形成了一个双顺反子操纵子，这表明他们形成了一个两个组件system.Within本补助金的建议，我们希望调查MsmS和同源激酶MA 0863和它们的作用，在调节corrinoid/甲基转移酶同工酶。一条线的调查将在这个原型血红素传感器激酶的血红素的配位结构的特点，特别是共价键的作用将被检查。初步结果表明MA 0863在其第二GAF结构域中也具有血红素结合。将研究氧化还原性质和轴向配位是否不同于在MSMS中发现的血红素。因此，将采用UV-vis、共振拉曼和磁性圆二色性光谱的组合。将使用氧化还原滴定实验测定氧化还原电位。虽然两种传感器具有高度的氨基酸序列同源性，但MA 0863的特殊之处在于它在其第二PAS结构域中编码琥珀密码子，该密码子在M中被翻译为氨基酸吡咯赖氨酸。食醋动物。吡咯赖氨酸是否作为常规氨基酸残基或具有特殊功能是本提案的另一个目标。从传感器激酶和可能的交叉调节的corrinoid/甲基转移酶同工酶的信号转导途径将进行研究。除了编码类咕啉/甲基转移酶同工酶的靶基因的转录调控外，我们还希望探索Msr调节子在信号转导过程中的功能。这将在传感器激酶和调节蛋白之间的蛋白质-蛋白质相互作用研究以及使用凝胶迁移率变动测定的DNA-调节蛋白相互作用研究的帮助下进行。总的来说，我们希望有助于了解古细菌的信号转导一般，但特别是产甲烷菌的生理。

The methyl sulfide methyltransferase associated Sensor MsmS (MA4561) from the methanogenic archaeon Methanosarcina acetivorans is a multidomain sensor protein consisting of alternating PAS and GAF domains joint to a C-terminal histidine kinase-like H_ATPase output domain. MsmS contains a covalently bound heme-cofactor in its second GAF domain. This heme is redox active and has the ability to ligate several external ligands including carbon monoxide and dimethyl sulfide. Upon oxidation of the heme cofactor autophosphorylation activity of the H_ATPase domain is induced suggesting that MsmS is a sensor kinase. With the help of a chromosomal knock out mutant of msmS we could further show that MsmS is involved in the cross-regulation of one of three gene encoding corrinoid/methyltransferase isozymes. The transcription of these genes was shown to be activated by upstream encoded regulators of the Msr family. One of these regulators is encoded downstream of msmS with which it forms a bicistronic operon suggesting that they form a two component system.Within this grant proposal we wish to investigate MsmS and the homologous kinase MA0863 and their role in regulating the corrinoid/methyltransferase isozymes. One line of investigation will characterize the coordination structure of the heme within this prototypical heme sensor kinase from Archaea and specifically the role of the covalent bond will be examined. Preliminary results indicated that MA0863 has also a heme bound in its second GAF domain. It will be investigated whether the redox properties and the axial coordination is different from the heme found in Msms. Therefore, a combination of UV-vis, resonance Raman und magnetic circular dichroism spectroscopy will be employed. Redox potentials will be determined using redox titration experiments. Although both sensors share high amino acid sequence homology, MA0863 is special in that it encodes an amber codon in its second PAS domain which is translated to the amino acid pyrrolysine in M. acetivorans. Whether pyrrolysine serves as a regular amino acid residue or holds a special function is another objective of this proposal. The signal transduction pathways from the sensor kinases and possible cross regulation of the corrinoid/methyltransferase isozymes will be investigated. Besides the transcriptional regulation of the target genes encoding the corrinoid/methyltransferase isozymes, we also wish to explore the function of the Msr regulators in the signal transduction process. This will be done with the help of protein-protein interaction studies between the sensor kinases and the regulator proteins and DNA-regulator interaction studies using gel mobility shift assays. Overall, we wish to contribute to the understanding of archaeal signal transduction in general but on methanogen physiology in particular.