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首页> 外文期刊>Biochemistry >Stereochemical course of enzymatic enolpyruvyl transfer and catalytic conformation of the active site revealed by the crystal structure of the fluorinated analogue of the reaction tetrahedral intermediate bound to the active site of the C115A mutant
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Stereochemical course of enzymatic enolpyruvyl transfer and catalytic conformation of the active site revealed by the crystal structure of the fluorinated analogue of the reaction tetrahedral intermediate bound to the active site of the C115A mutant

机译：反应四面体中间体的氟化类似物的晶体结构结合到C115A突变体的活性位点上，揭示了酶促烯醇式丙酮酸转移的立体化学过程和活性位点的催化构象

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摘要

MurA (UDP-GlcNAc enolpyruvyl transferase), the first enzyme in bacterial peptidoglycan biosynthesis, catalyzes the enolpyruvyl transfer from phosphoenolpyruvate (PEP) to the 3'-OH of UDP-GlcNAc by an addition-elimination mechanism that proceeds through a tetrahedral ketal intermediate. The crystal structure of the Cys115-to-Ala (C115A) mutant of Escherichia coli MurA complexed with a fluoro analogue of the tetrahedral intermediate revealed the absolute configuration of the adduct and the stereochemical course of the reaction, The fluorinated adduct was generated in a preincubation of wild-type MurA with (Z)-3-fluorophasphoenolpyruvate (FPEP) and UDP-GlcNAc and purified after enzyme denaturation. The fluorine substituent stabilizes the tetrahedral intermediate toward decomposition by a factor of 10(4)-10(6), facilitating manipulation of the adduct. The C115A mutant of MurA was utilized to avoid the microheterogeneity that arises in the wild-type MurA from the attack of Cys115 on C-2 of FPEP in competition with the formation of the fluorinated adduct. The crystal structure of the complex was determined to 2.8 Angstrom resolution, and the absolute configuration at C-2 of the adduct was found to be 2R, Thus, addition elf the 3'-OH of UDP-GlcNAc is to the 2-si face of FPEP, corresponding to the 2-re face of PEP. Given the previous observation that, in D2O the addition of D+ to C-3 of PEP proceeds from the 2-si face [Kim, D. H., Lees, W. J., and Walsh, C. T, (1995) J. Am. Chem. Sec. 117, 6380-6381], the addition across the double bond of PEP is anti, Also, because the overall stereochemical course has been shown to be either anti/syn or syn/anti [Lees, W. J., and Walsh, C. T. (1995) J. Am. Chem. Sec. 117, 7329-7337], it now follows that the stereochemistry of elimination of H+ from C-3 and Pi from C-2 of the tetrahedral intermediate of the reaction is syn. [References: 25]
机译：细菌肽聚糖生物合成中的第一个酶MurA（UDP-GlcNAc烯醇丙酮酸转移酶），通过四面体缩酮中间体进行加成消除机理，催化烯醇丙酮酸从磷酸烯醇丙酮酸（PEP）转移到UDP-GlcNAc的3'-OH。与四面体中间体的氟类似物复合的大肠杆菌MurA的Cys115-Ala（C115A）突变体的晶体结构显示了加合物的绝对构型和反应的立体化学过程。氟化的加合物在预温育中生成（Z）-3-氟代磷酸烯醇丙酮酸（FPEP）和UDP-GlcNAc制备野生型MurA，并在酶变性后纯化。氟取代基使四面体中间产物稳定分解，分解系数为10（4）-10（6），有利于加合物的操作。利用Mur115的C115A突变体避免了Cys115对FPEP C-2的攻击，与氟化加合物的形成竞争，从而在野生型MurA中产生了微异质性。测定该络合物的晶体结构至2.8埃的分辨率，并且发现加合物的C-2的绝对构型为2R，因此，将UDP-GlcNAc的3'-OH加成到2-si面上对应于PEP的2面。鉴于先前的观察，在D2O中，将D +添加到PEP的C-3中是从2-si面开始的[Kim，D. H.，Lees，W. J.和Walsh，C. T，（1995）J. Am。化学秒117，6380-6381]，PEP双键上的加成是抗的。而且，因为已显示总体立体化学过程是抗/ syn或syn / anti [Lees，WJ和Walsh，CT（1995）。 J.上午化学秒117，7329-7337]，现在得出反应四面体中间体从C-3除去H +和从C-2除去Pi的立体化学是顺式。 [参考：25]

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