Core H: Microbiology

核心 H：微生物学

• 批准号: 7980205
• 负责人: John E. Cronan
• 金额: $ 78.75万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-20 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7980205
• 关键词: Applied Genetics; Biochemical; Biological Assay; Core Protein; Coupled; DNA; DNA Polymerase I; DNA Repair; DNA biosynthesis; DNA-Directed DNA Polymerase; Data; Dipeptides; Effectiveness; Enzymes; Escherichia coli; Evaluation; Fractionation; Genes; Genetic; Genome; Genomics; In Vitro; Knock-out; Mass Spectrum Analysis; Microbial Genetics; Microbiology; Mutagens; Nobel Prize; Phenotype; Physiological; Plasmids; Polymerase; Preparation; Production; Proteins; Racemases; Reaction; Research Infrastructure; Role; Structure; Subgroup; arginyllysine; bacterial genetics; base; enolase; epimerase; genetic analysis; in vivo; member; metabolomics; mutant; overexpression; programs

IN VITRO vs. IN VIVO FUNCTION The most reliable approach for establishing the in vivo function of an enzyme is by genetics coupled with biochemical analyses. Numerous examples demonstrate that the physiological roles assigned to enzymes based on in vitro reactions they catalyze are incorrect. In perhaps tne most famous example, Arthur Kornberg discovered DNA polymerase I which was thought to be the sole E. coli DNA polymerase and, therefore, must be responsible for DNA replication [2]. However, mutants lacking DNA polymerase I grew and made DNA normally; hence, DNA polymerase I could not be the replicatlve polymerase [3, 4]. The replicatlve polymerase later was shown to be DNA polymerase 111, a much more complicated enzyme that, given the reaction conditions and template used for DNA polymerase I, is virtually inactive. However, mutants of polymerase I are super-sensitive to both UV and mutagens, showing that the in vivo role of polymerase I is DNA repair [5]. Kornberg desen/ed the Nobel Prize, but the fact remains that the physiological role of DNA polymerase I was in error due to the lack of genetics. A less famous but immediately relevant example of the in vivo ambiguity of an In vitro assigned function is provided by the computationally predicted and experimentally verified assignments of the N-succinyl Arg/Lys racemase [6] and L-Ala-D/L-Phe dipeptide epimerase [7] functions to members the MLE subgroup of the enolase superfamily. In both examples, Jacobson (Computation Core) predicted substrate promiscuity that was confirmed by enzymatic assays (EN Bridging Project). However, for both enzymes, the identity of the in vivo substrate as well as the physiological importance of the reaction is unknown. The Microbiology Core will apply genetic analyses and metabolomics to determine the in vivo roles of enzymes for which the In vitro functions are predicted by the Computation Core and verified by the Bridging Projects. Enzymes that emerge from the bottom of the funnel of Figure 1 in the Program Summary may be active with one, a few, or many related substrates with varying catalytic efficiencies. Which substrate(s) do they use in vivo? A combination of bacterial genetics, phenotypic characterization, and metabolite analysis will enable the physiological roles of the EFI targets to be evaluated and assigned.

体外与体内功能 建立酶的体内功能的最可靠方法是通过遗传学以及生化分析。许多例子表明，基于催化的体外反应分配给酶的生理作用是不正确的。在也许最著名的例子中，亚瑟·科恩伯格（Arthur Kornberg 因此，必须负责DNA复制[2]。然而，缺乏DNA聚合酶I的突变体生长并正常制成DNA。因此，DNA聚合酶I不能成为复制聚合酶[3，4]。后来的复制聚合酶被证明是DNA聚合酶111，这是一种更为复杂的酶，鉴于该酶 用于DNA聚合酶I的反应条件和模板实际上是非活性的。但是，聚合酶I的突变体对紫外线和诱变剂都非常敏感，表明聚合酶I的体内作用是DNA修复[5]。 Kornberg Desen/Ed诺贝尔奖，但事实仍然是，由于缺乏遗传学，DNA聚合酶I的生理作用是错误的。 在计算预测和实验验证的N-核酸ARG/LYS RASEMASE [6]和L-ALA-D/L-PHE Dipeptide Epimase酶的MLE子集团的MLE子组中提供了一个不太知名但立即相关的体内歧义歧义的示例[6]和实验验证的分配[6] 烯醇酶超家族。在这两个示例中，Jacobson（计算核心）都预测了通过酶试验（EN BRIDGING项目）证实的底物滥交。但是，对于这两种酶，体内底物的身份以及反应的生理重要性尚不清楚。 微生物核心将采用遗传分析和代谢组学来确定由计算核心预测体外功能的酶的体内作用，并由桥接项目验证。从程序摘要中从图1的漏斗底部出现的酶可能具有一个，几个或许多相关底物具有不同催化效率的酶。哪个底物是 他们在体内使用吗？细菌遗传学，表型表征和代谢产物分析的结合将使EFI靶标的生理作用得到评估和分配。