Core H: Microbiology

体外功能与体内功能 确定酶在体内的功能最可靠的方法是通过遗传学和生化分析相结合的方法。大量的例子表明，根据酶催化的体外反应分配给酶的生理作用是不正确的。在一个最著名的例子中，Arthur Kornberg发现了DNA聚合酶I，它被认为是唯一的大肠杆菌DNA聚合酶， 因此，必须负责DNA复制[2]。然而，缺乏DNA聚合酶I的突变体正常生长和合成DNA，因此DNA聚合酶I不可能是复制聚合酶[3，4]。复制聚合酶后来被证明是DNA聚合酶111，一种复杂得多的酶，鉴于 用于DNA聚合酶I的反应条件和模板实际上是无效的。然而，聚合酶I的突变体对紫外线和诱变剂都超敏感，这表明聚合酶I在体内的作用是DNA修复[5]。科恩伯格获得了诺贝尔奖，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。 通过计算预测和实验验证N-琥珀酰精氨酸/赖氨酸外消旋酶[6]和L-丙氨酸-D/L-苯丙氨酸二肽异构体酶[7]的功能被分配给 烯醇化酶超家族。在这两个例子中，雅各布森(计算核心)预测了底物的混杂，这一预测得到了酶分析(EN桥接项目)的证实。然而，对于这两种酶来说，体内底物的一致性以及反应的生理重要性尚不清楚。 微生物学核心将应用遗传分析和代谢组学来确定酶在体内的作用，这些酶的体外功能由计算核心预测，并由桥梁项目验证。在程序概要中，从图1漏斗底部出现的酶可能与一个、几个或许多具有不同催化效率的相关底物一起发挥活性。哪种底物(S)做的 他们在活体内使用？细菌遗传学、表型特征和代谢物分析的结合将使EFI靶标的生理作用得到评估和分配。

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.