B12 Biosysthesis and Anaerobic Metabolism in Salmonella

描述（由申请人提供）：维生素B12（又名氰钴胺素）是人类必需的营养素，但它只能由原核生物合成。维生素的生物活性形式被称为辅酶B12（又名腺苷钴胺素，AdoCbl）。将维生素转化为辅酶形式需要在B12的钴原子和ATP的腺苷基之间形成独特的有机金属键。我们试图了解形成辅酶的Co-C键的途径步骤的分子细节。反应是非常不利的，但我们已经大大提高了我们对如何克服反对反应的能量势垒的理解。研究了人致病菌肠沙门氏菌CobA腺苷转移酶的催化作用机制。我们最近在肠球菌中发现了一种新型的腺苷转移酶（EutT），它可能通过一种涉及尚未确定的金属中心的机制催化该反应。我们建议继续我们对CobA的机制分析，并启动EutT酶的生化，遗传和结构表征。我们还建议研究古细菌特有的两种新的途径酶。这些酶，CobY和cbi，分别参与了途径组装的后期步骤和从环境中回收预先形成的前体。在细菌中，古细菌CobY酶的对应物（肠链球菌中的CobU）在进化上与CobY无关，而且更复杂。我们有一个独特的机会来剖析两种不同的催化机制，它们必须在不同的选择压力下进化。更好地了解细菌CobU酶是有意义的，因为这种酶是B12从头合成和从环境中回收前体所必需的，因此它代表了开发新抗生素的潜在目标。CbiS酶代表了前体回收的新途径。cbi的吸引人之处在于，它只在生活在公元前100年的古细菌中被发现。cbi实际上是两种酶合二为一，它的生化、结构和遗传分析将提供有关蛋白质热稳定性的宝贵信息，以及细胞在极端环境中使用的策略，以稳定通路的关键步骤。最后，我们将开始剖析下配基的生物合成途径。一种途径主要是非酶促的，而另一种途径是零碎的。

DESCRIPTION (provided by applicant): Vitamin B12 (aka cyanocobalamin) is an essential nutrient to humans but it is only synthesized by prokaryotes. The biologically active form of the vitamin is known as coenzyme B12 (aka adenosylcobalamin, AdoCbl). The conversion of the vitamin to the coenzymic form requires the formation of a unique organometallic bond between the cobalt atom of B12 and the adenosyl group from ATP. We seek to understand the molecular details of the step of the pathway that forms the Co-C bond of the coenzyme. The reaction is very unfavorable but we have substantially improved our understanding of how the energy barrier that opposes the reaction is overcome. We have studied the mechanism of catalysis of the CobA adenosyltrasnferase enzyme of the human pathogen Salmonella enterica. We recently discovered a new type of adenosyltransferase (EutT) in S. enterica that is likely to catalyze the reaction via a mechanism that involves an as-yet-undefined metal center. We propose to continue our mechanistic analysis of CobA and to initiate the biochemical, genetic and structural characterization of the EutT enzyme. We also propose to study two new enzymes of the pathway that are unique to archaea. These enzymes, CobY and CbiS, are involved in the late steps of the assembly of the pathway and in the salvaging of preformed precursors from the environment, respectively. In bacteria, the counterpart for the archaeal CobY enzyme (CobU in S. enterica) is evolutionarily unrelated to CobY, and is more complex. We have a unique opportunity to dissect two different mechanisms of catalysis that must have evolved in response to different selective pressures. A better understanding of the bacterial CobU enzyme is of interest since this enzyme is needed for de novo synthesis of B12 and for salvaging precursors from the environment, hence it represents a potential target for the development of new antibiotics. The CbiS enzyme represents a new pathway for precursor salvaging. What is attractive about CbiS is that it has only been found in archaea living in >100¿C. CbiS is actually two enzymes in one, and its biochemical, structural and genetic analyses will yield valuable information about thermostability of proteins as well as strategies used by cells occupying extreme environments to stabilize key steps of the pathway. Lastly, we will begin to dissect the biosynthetic pathways for the lower ligand base. One pathway is largely non-enzymatic while the other one is a piecemeal pathway.