The bacterial class IIIb adenylate cyclase CyaC-Sm of Sinorhizobium meliloti: Redox sensing by a heme B binding membrane anchor

原核和真核腺苷酸环化酶：具有感觉功能的膜锚？从ATP产生第二信使环AMP的腺苷酸环化酶（AC）存在于所有类别的生命中（真核生物、细菌和古细菌）。环AMP在调节细胞功能方面具有广泛的意义。AC酶分为六类，其中最具代表性的AC酶是III类，它涵盖了大多数真核生物和细菌的AC酶。这类ac在域组成、信号感知、调节和整体功能方面表现出很大的差异。该提案旨在了解Sinorhizobium meliloti （IIIb类AC）的CyaC-Sm的功能及其六螺旋（6-TM）膜锚点在氧化还原传感中的作用。6-TM膜锚在真核生物（包括哺乳动物）和一些细菌III类ACs中是保守的。这种设计的细菌ac尚未在分子细节上进行表征。了解CyaC-Sm可能对评估膜锚及其在真核III类ACs中的潜在感觉作用有影响。前期工作表明CyaC-Sm在膜锚中含有醌反应性血红素B。呼吸作用和醌类被认为可以氧化铁氧还蛋白型结构域中的血红素B和铁硫簇，从而控制催化结构域的二聚化和活性。具有醌/血红素位点的膜锚和附近的FeS簇在结构和功能方面与已被充分表征的呼吸富马酸还原酶的结构域具有显著的相似性。通过纯化的CyaC-Sm，以及在大肠杆菌中表达的野生型和突变型CyaC-Sm， SMc01816的组分和连接氧化还原酶（SMc01816）在控制AC活性中的作用将得到表征。总体而言，该研究旨在表征CyaC-Sm作为一种新颖的，实际上是第一个，氧化还原控制的III类腺苷酸环化酶的功能和分子细节。这项工作可能具有更广泛的意义，因为它赋予了AC蛋白的六螺旋TM结构域一种感觉作用，这种作用也被讨论用于膜分隔的真核AC。本研究未对CyaC-Sm在香菇中的生理作用进行分析。然而，有初步迹象表明CyaC-Sm与有氧呼吸和反硝化有关。

Pro- and eukaryotic adenylate cyclases: Membrane anchors with sensory function?Adenylate cyclases (AC) producing the second messenger cyclic AMP from ATP are present in all classes of life (eukarya, bacteria and archaea). Cyclic AMP has a broad significance in regulating cellular functions. The AC enzymes are grouped in six classes, and the best characterized ACs are members of class III which covers most eukaryotic and bacterial ACs. The ACs of this class show a large variation in domain composition, signal perception, regulation and overall function. The proposal aims at understanding the function of CyaC-Sm of Sinorhizobium meliloti, a class IIIb AC, and the role of its hexa-helical (6-TM) membrane anchor in redox sensing. A 6-TM membrane anchor is conserved in the eukaryotic (including mammalian) and some bacterial class III ACs. Bacterial ACs of this design have not been characterized in molecular detail. Understanding CyaC-Sm may have an impact on assessing the membrane anchor and its potential sensory role in eukaryotic class III ACs. Preparatory work suggests that CyaC-Sm contains a quinone reactive heme B in the membrane anchor. Respiration and quinones are supposed to oxidize the heme B and an iron-sulfur cluster in a ferredoxin-type domain and, as a result, to control dimerization and activity of the catalytic domain. The membrane anchor with the quinone/heme site and the near-by FeS cluster show a remarkable similarity to domains of the well-characterized respiratory fumarate reductase in structural and functional terms. The role of the components and of a linked oxidoreductase (SMc01816) in controlling AC activity will be charac-terized with purified CyaC-Sm, and by the use of wild-type and mutant CyaC-Sm expressed in E. coli. Overall, the study aims at characterizing function and molecular details of CyaC-Sm as a novel, actually the first, redox-controlled class III adenylate cyclase. The work might be of broader significance by assigning the hexa-helical TM domain of AC proteins a sensory role which is also discussed for membrane-delimited eukaryotic ACs. The physiological role of CyaC-Sm in S. meliloti is not analyzed in the proposal. However, there are first indications that CyaC-Sm is linked to aerobic respiration and to denitrification.