Skarzynski T, Kim D H, Lees W J, Walsh C T, Duncan K
Glaxo Wellcome Research and Development, Medicines Research Centre, Stevenage, Hertfordshire, United Kingdom SG1 2NY.
Biochemistry. 1998 Feb 24;37(8):2572-7. doi: 10.1021/bi9722608.
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-fluorophosphoenolpyruvate (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 A resolution, and the absolute configuration at C-2 of the adduct was found to be 2R. Thus, addition of 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. Soc. 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. Soc. 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.
MurA（尿苷二磷酸-N-乙酰葡糖胺烯醇丙酮酸转移酶）是细菌肽聚糖生物合成过程中的首个酶，它通过一种加成-消除机制催化磷酸烯醇丙酮酸（PEP）的烯醇丙酮酸基转移至尿苷二磷酸-N-乙酰葡糖胺（UDP-GlcNAc）的3'-OH上，该机制通过一个四面体缩酮中间体进行。大肠杆菌MurA的半胱氨酸115突变为丙氨酸（C115A）的突变体与四面体中间体的氟代类似物形成的复合物的晶体结构揭示了加合物的绝对构型以及反应的立体化学过程。将野生型MurA与（Z）-3-氟磷酸烯醇丙酮酸（FPEP）和UDP-GlcNAc预孵育生成氟化加合物，经酶变性后进行纯化。氟取代基使四面体中间体的稳定性提高了10^4 - 10^6倍，便于对加合物进行操作。利用MurA的C115A突变体避免了野生型MurA中因半胱氨酸115对FPEP C-2位的进攻与氟化加合物形成竞争而产生微小异质性。复合物的晶体结构解析到2.8 Å分辨率，发现加合物C-2位的绝对构型为2R。因此，UDP-GlcNAc的3'-OH加到FPEP的2-si面，对应于PEP的2-re面。鉴于之前的观察结果，即在重水中，D+加到PEP的C-3位是从2-si面进行的[Kim, D. H., Lees, W. J., and Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 6380 - 6381]，PEP双键上的加成是反式的。此外，由于整体立体化学过程已被证明是反式/顺式或顺式/反式[Lees, W. J., and Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 7329 - 7337]，现在可以得出，反应四面体中间体中从C-3位消除H+以及从C-2位消除磷酸根离子的立体化学是顺式的。
