Evolution of enzymatic activity in the tautomerase superfamily: Mechanistic and structural consequences of the L8R mutation in 4-oxalocrotonate Tautomerase
Evolution of enzymatic activity in the tautomerase superfamily: Mechanistic and structural consequences of the L8R mutation in 4-oxalocrotonate Tautomerase
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DOI：
10.1021/bi0600603
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发表时间：
2006-06-27
期刊：
影响因子：
2.9
通讯作者：
Whitman, Christian P.
中科院分区：
文献类型：
--
作者：
Poelarends, Gerrit J.;Almrud, Jeffrey J.;Whitman, Christian P.
4-草糖醛酸互变异构体酶（4-OT）和反式3-氯丙烯酸脱卤酶（CaaD）是互变异构体酶超家族的成员，这是一组结构上同源的蛋白质，具有- α - β折叠和催化氨基末端脯氨酸。来自恶臭假单胞菌mt-2的4-OT在芳香烃的分解代谢途径中通过二烯醇中间体2-羟基酸酯催化2-氧-4-己烯二酸转化为2-氧-3-己烯二酸。来自pavonaceae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-氯丙烯酸酯水解脱卤。这两种反应都可能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低CaaD活性的原因。4-OT中的两个活性位点，Leu-8和Ile-52，现在已经突变为CaaD， RArg-8和RGlu-52中的位置保守和催化残基。L8R和L8R/I52E突变体的CaaD活性提高（k(cat)/ k -m分别增加50倍和32倍），4-OT活性降低（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是由于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减少了23倍。在L8R-4-OT活性位点存在额外的精氨酸残基不会改变野生型Pro-1氨基的pK(a) （6.5 +/- 0.1 vs 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的CaaD活性增强可能是由于额外的精氨酸残基可以参与底物结合和/或稳定假定的烯二酸中间体。研究结果还表明，在互变异构酶超家族中，新功能的进化可能相当容易，只需要一些战略性的活性位点突变。
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, RArg-8, and RGlu-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.