Lambert Peter M, Nakata Paul A
USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX, 77030-2600.
PLoS One. 2016 Sep 19;11(9):e0163294. doi: 10.1371/journal.pone.0163294. eCollection 2016.
Oxalic acid is produced by a variety of organisms ranging from simple microbes to complex animals. This acid has been proposed to fulfill various physiological and pathological functions which vary between organisms. In bacteria from the Burkholderia genus, oxalate secretion has been shown to be quorum sensing dependent and to support pathogenicity and cell viability. In light of the critical roles of oxalate in Burkholderia as well as other organisms, it is surprising that our understanding of how this simple dicarboxylate is biosynthesized remains incomplete. Here we report the expression, purification, and partial characterization of the first intact bacterial oxalate biosynthetic enzyme, Obc1, from B. mallei. An N-terminal His-tagged Bmobc1 was cloned into pDUET, expressed in E. coli BLR (DE3), and the recombinant enzyme purified by affinity chromatography. Oxalate biosynthetic enzyme assays coupled with HPLC analysis revealed that BmObc1 catalyzed the biosynthesis of oxalate, acetoacetate, and free CoA from oxaloacetate and a short chain acyl-CoA following Michaelis-Menten kinetics. Optimal enzyme activity was measured at pH 8.0 and a temperature around 44°C. Kinetic analysis conducted under conditions of saturating acetyl-CoA and varying oxaloacetate concentrations resulted in a calculated Km value for oxaloacetate of 94.3± 9.2 μM (mean ± SE). Under conditions of saturating oxaloacetate concentration and varying acyl-CoA (acetyl- or propionyl-CoA) concentrations kinetic analysis generated a calculated Km value of 26.8 ± 2.3 μM (mean ± SE) for acetyl-CoA and 104.4 ± 12.7 μM for propionyl-CoA. The significantly lower Km for acetyl-CoA suggests that it is strongly favored as a substrate over propionyl-CoA.
草酸由从简单微生物到复杂动物等多种生物体产生。有人提出这种酸具有多种生理和病理功能，这些功能在不同生物体之间有所不同。在伯克霍尔德菌属的细菌中，草酸盐分泌已被证明依赖群体感应，并支持致病性和细胞活力。鉴于草酸盐在伯克霍尔德菌以及其他生物体中的关键作用，令人惊讶的是，我们对这种简单二羧酸的生物合成方式的理解仍然不完整。在这里，我们报告了来自鼻疽伯克霍尔德菌的首个完整细菌草酸生物合成酶Obc1的表达、纯化及部分特性分析。将N端带有His标签的Bmobc1克隆到pDUET中，在大肠杆菌BLR(DE3)中表达，并用亲和色谱法纯化重组酶。草酸生物合成酶测定结合HPLC分析表明，BmObc1按照米氏动力学催化从草酰乙酸和短链酰基辅酶A生物合成草酸、乙酰乙酸和游离辅酶A。在pH 8.0和44°C左右的温度下测得最佳酶活性。在乙酰辅酶A饱和且草酰乙酸浓度变化的条件下进行动力学分析，得出草酰乙酸的计算Km值为94.3±9.2μM(平均值±标准误)。在草酰乙酸浓度饱和且酰基辅酶A(乙酰或丙酰辅酶A)浓度变化的条件下进行动力学分析，得出乙酰辅酶A的计算Km值为26.8±2.3μM(平均值±标准误)，丙酰辅酶A的计算Km值为104.4±12.7μM。乙酰辅酶A的Km值显著更低，这表明它作为底物比丙酰辅酶A更受青睐。
