B12 Biosysthesis and Anaerobic Metabolism in Salmonella

描述（由申请人提供）：维生素B12（又名氰钴胺）是人体必需的营养素，但仅由原核生物合成。维生素的生物活性形式被称为辅酶B12（aka adenosylcobalamin，CACBl）。维生素转化为辅酶形式需要在B12的钴原子和ATP的腺苷基团之间形成独特的有机金属键。我们试图了解形成辅酶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的理解，如何克服能量障碍，反对反应。我们研究了人类病原体肠道沙门氏菌的CobA腺苷转移酶的催化机制。我们最近在S.肠，可能通过涉及尚未定义的金属中心的机制催化反应。我们建议继续我们的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2个新的酶的途径，是独特的古细菌。这些酶，CobY和CbiS，分别参与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，与古细菌CobY酶（S. enterica）在进化上与CobY无关，并且更复杂。我们有一个独特的机会来剖析2种不同的催化机制，它们必须在不同的选择压力下进化。对细菌CobU酶的更好理解是令人感兴趣的，因为这种酶是从头合成B12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靶标。CbiS酶代表了前体回收的新途径。CbiS的吸引人之处在于，它只在生活在>100年的古生菌中被发现。C. CbiS实际上是2合1的酶，它的生物化学、结构和遗传分析将产生有关蛋白质热稳定性的有价值的信息，以及占据极端环境的细胞稳定途径关键步骤的策略。最后，我们将开始剖析生物合成途径为较低的配体基地。1个途径主要是非酶的，而另一个是零碎的途径。

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 2 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 2 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 2 enzymes in 1, 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. 1 pathway is largely non-enzymatic while the other one is a piecemeal pathway.