Instituto de Investigaciones Químico Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Edificio B-3, Ciudad Universitaria, 58030 Morelia, Michoacán, Mexico.
World J Microbiol Biotechnol. 2012 Mar;28(3):1185-91. doi: 10.1007/s11274-011-0921-1. Epub 2011 Oct 20.
Pseudomonas aeruginosa is a versatile bacterium that can grow using citronellol or leucine as sole carbon source. For both compounds the degradation pathways converge at the key enzyme 3-methylcrotonyl coenzyme-A carboxylase (MCCase). This enzyme is a complex formed by two subunits (α and β), encoded by the liuD and liuB genes, respectively; both are essential for enzyme function. Previously, both subunits had been separately expressed and then the complex re-constituted, however this methodology is laborious and produces low yield of active enzyme. In this work, the MCCase subunits were co-expressed in the same plasmid and purified in one step by affinity chromatography using the LiuD-His tag protein, interacting with the LiuB-S tag recombinant protein. The purified enzyme lost most of the activity within few hours of storage. The co-expressed subunits formed an (αβ)(4) complex that suffered a modification of its oligomerization state after storage, which probably contributed to the loss on activity observed. The recombinant MCCase enzyme presented optimum pH and temperature values of 9.0 and 30º C, respectively. Functionally, MCCase showed Michaelian kinetics behavior with a K(m) for its substrate and V(max) of 168 μM and 430 nmoles mg(-1)min(-1), respectively. The results suggest that the co-expression and co-purification of the subunits is a suitable procedure to obtain the active complex of the MCCase from Pseudomonas aeruginosa in a single step.
铜绿假单胞菌是一种多功能细菌，可以以香茅醇或亮氨酸作为唯一碳源生长。对于这两种化合物，降解途径都集中在关键酶 3-甲基巴豆酰辅酶 A 羧化酶（MCCase）上。该酶是由两个亚基（α和β）组成的复合物，分别由 liuD 和 liuB 基因编码；两者对于酶功能都是必需的。以前，已经分别表达了这两个亚基，然后再重新构建复合物，但这种方法很繁琐，并且产生的活性酶产量低。在这项工作中，MCCase 的亚基在同一个质粒中共表达，并通过使用 LiuD-His 标记蛋白的亲和层析一步法进行纯化，该蛋白与 LiuB-S 标记的重组蛋白相互作用。纯化的酶在储存数小时后会失去大部分活性。共表达的亚基形成了一个（αβ）（4）复合物，在储存后其寡聚化状态发生了修饰，这可能是导致观察到的活性丧失的原因。重组 MCCase 酶的最适 pH 和温度值分别为 9.0 和 30°C。在功能上，MCCase 表现出米氏动力学行为，其底物的 K(m)和 V(max)分别为 168 μM 和 430 nmoles mg(-1)min(-1)。结果表明，亚基的共表达和共纯化是从铜绿假单胞菌中一步获得 MCCase 活性复合物的合适方法。
