Poelarends Gerrit J, Almrud Jeffrey J, Serrano Hector, Darty Joseph E, Johnson William H, Hackert Marvin L, Whitman Christian P
Division of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry, The University of Texas, Austin, Texas 78712-1074, USA.
Biochemistry. 2006 Jun 27;45(25):7700-8. doi: 10.1021/bi0600603.
4-Oxalocrotonate tautomerase (4-OT) and trans-3-chloroacrylic acid dehalogenase (CaaD) are members of the tautomerase superfamily, a group of structurally homologous proteins that share a beta-alpha-beta fold and a catalytic amino-terminal proline. 4-OT, from Pseudomonas putida mt-2, catalyzes the conversion of 2-oxo-4-hexenedioate to 2-oxo-3-hexenedioate through the dienol intermediate 2-hydroxymuconate in a catabolic pathway for aromatic hydrocarbons. CaaD, from Pseudomonas pavonaceae 170, catalyzes the hydrolytic dehalogenation of trans-3-chloroacrylate in the trans-1,3-dichloropropene degradation pathway. Both reactions may involve an arginine-stabilized enediolate intermediate, a capability that may partially account for the low-level CaaD activity of 4-OT. Two active-site residues in 4-OT, Leu-8 and Ile-52, have now been mutated to the positionally conserved and catalytic ones in CaaD, alphaArg-8, and alphaGlu-52. The L8R and L8R/I52E mutants show improved CaaD activity (50- and 32-fold increases in k(cat)/K(m), respectively) and diminished 4-OT activity (5- and 1700-fold decreases in k(cat)/K(m), respectively). The increased efficiency of L8R-4-OT for the CaaD reaction stems primarily from an 8.8-fold increase in k(cat), whereas that of the L8R/I52E mutant is due largely to a 23-fold decrease in K(m). The presence of the additional arginine residue in the active site of L8R-4-OT does not alter the pK(a) of the Pro-1 amino group from that measured for the wild type (6.5 +/- 0.1 versus 6.4 +/- 0.2). Moreover, the crystal structure of L8R-4-OT is comparable to that of the wild type. Hence, the enhanced CaaD activity of L8R-4-OT is likely due to the additional arginine residue that can participate in substrate binding and/or stabilization of the putative enediolate intermediate. The results also suggest that the evolution of new functions within the tautomerase superfamily could be quite facile, requiring only a few strategically placed active-site mutations.
4-草酰巴豆酸互变异构酶（4-OT）和反式-3-氯丙烯酸脱卤酶（CaaD）是互变异构酶超家族的成员，该超家族是一组结构同源的蛋白质，具有β-α-β折叠和催化性氨基末端脯氨酸。来自恶臭假单胞菌mt-2的4-OT在芳烃的分解代谢途径中，通过二烯醇中间体2-羟基粘康酸催化2-氧代-4-己烯二酸酯转化为2-氧代-3-己烯二酸酯。来自孔雀假单胞菌170的CaaD在反式-1,3-二氯丙烯降解途径中催化反式-3-氯丙烯酸酯的水解脱卤反应。这两个反应可能都涉及精氨酸稳定的烯二醇中间体，这种能力可能部分解释了4-OT的低水平CaaD活性。4-OT中的两个活性位点残基Leu-8和Ile-52，现已突变为CaaD中位置保守且具有催化作用的残基αArg-8和αGlu-52。L8R和L8R/I52E突变体显示出改善的CaaD活性（k(cat)/K(m)分别增加50倍和32倍）以及降低的4-OT活性（k(cat)/K(m)分别降低5倍和1700倍）。L8R-4-OT对CaaD反应效率的提高主要源于k(cat)增加了8.8倍，而L8R/I52E突变体的效率提高主要是由于K(m)降低了23倍。L8R-4-OT活性位点中额外精氨酸残基的存在并未改变Pro-1氨基的pK(a)，与野生型测量值相比（分别为6.5±0.1和6.4±0.2）。此外，L8R-4-OT的晶体结构与野生型相当。因此，L8R-4-OT增强的CaaD活性可能归因于额外的精氨酸残基，它可以参与底物结合和/或假定烯二醇中间体的稳定。结果还表明，互变异构酶超家族内新功能的进化可能相当容易，只需要在活性位点进行一些策略性的突变。
