Kinetic and structural characterization of dihydrofolate reductase from Streptococcus pneumoniae.
Kinetic and structural characterization of dihydrofolate reductase from Streptococcus pneumoniae.
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DOI：
10.1021/bi901614m
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发表时间：
2010-01-12
期刊：
影响因子：
2.9
通讯作者：
Benkovic SJ
中科院分区：
文献类型：
--
作者：
Lee J;Yennawar NH;Gam J;Benkovic SJ
与二氢叶酸还原酶(DHFR)相关的耐药性已成为细菌感染治疗中的一个关键问题。为了了解病原菌来源的耐药二氢叶酸还原酶(DHFR)的机制，我们首次报道了肺炎链球菌DHFR(SpDHFR)的动力学特征及其X射线结构。本研究表明，spDHFR的动力学性质与E.coliDHFR有显著不同。产物(四氢叶酸)解离步骤，即在大肠杆菌DHFR中的限速步骤，在spDHFR中被显着加速，因此氢化物转移或之前的步骤是限速步骤。将该酶与突变体spDHFR(Sp9)的结合参数进行比较，证实spDHFR中的Leu100残基是甲氧苄氨嘧啶(TMP)抗性的关键元件。在kcat中，稳态动力学表现出pH依赖性，这促使我们阐明了新的催化残基(His33)在spDHFR活性中心的作用。SP9突变体与NADPH和甲氨蝶呤的复合体的结构数据证实了His33参与了水分子、Thr119的羟基和Glu30的羧酸根离子的氢键网络。对DHFR超家族的序列分析表明，His残基是该位置的主要氨基酸成分，主要存在于病原菌DHFRs中。Val100到Leu的突变显示亮氨酸侧链与Ile8和Phe34的侧链发生空间碰撞，使甲氧苄啶与Leu100 dhfr结合较弱的合理化。了解特定氨基酸在活性部位的作用，再加上详细的结构分析，将为我们提供如何更好地设计针对耐药病原菌DHFRs的抑制剂。
Drug resistance associated with dihydrofolate reductase (DHFR) has emerged as a critical issue in the treatment of bacterial infections. In our efforts to understand the mechanism of a drug-resistant dihydrofolate reductase (DHFR) from a pathogenic bacterial source, we report the first kinetic characterization of Streptococcus pneumoniae DHFR (spDHFR) along with its X-ray structure. This study revealed that the kinetic properties of spDHFR were significantly different from E. coli DHFR. The product (tetrahydrofolate) dissociation step that is the rate limiting step in the E. coli DHFR is significantly accelerated in spDHFR so that hydride transfer or a preceding step is rate limiting. Comparison of the binding parameters of this enzyme to a mutant spDHFR (Sp9) confirmed that the Leu100 residue in spDHFR is the critical element for the trimethoprim (TMP) resistance. Steady-state kinetics exhibited a pH dependence in kcat, which prompted us to elucidate the role of the new catalytic residue (His33) in the active site of spDHFR. Structural data of the Sp9 mutant in complex with NADPH and methotrexate confirmed the participation of His33 in a hydrogen bonding network involving a water molecule, the hydroxyl group of Thr119, and carboxylate ion of Glu30. Sequence analysis of the DHFR superfamily revealed that the His residue is the major amino acid component at this position and is found mostly in pathogenic bacterial DHFRs. A mutation of Val100 to Leu demonstrated a steric clash of the leucine side chain with the side chains of Ile8 and Phe34, rationalizing weaker binding of trimethoprim to Leu100 DHFR. Understanding the role of specific amino acids in the active site coupled with detailed structural analysis will inform us on how to better design inhibitors targeting drug resistant pathogenic bacterial DHFRs.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
56.9
作者：
HOWELL, EE;VILLAFRANCA, JE;KRAUT, J
通讯作者：
KRAUT, J
影响因子：
5.6
作者：
Li, RB;Sirawaraporn, R;Hol, WGJ
通讯作者：
Hol, WGJ
DOI：
10.1107/s0907444998003254
发表时间：
1998-09-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Brunger, AT;Adams, PD;Warren, GL
通讯作者：
Warren, GL
DOI：
10.1073/pnas.120163297
发表时间：
2000-06-06
影响因子：
11.1
作者：
Datsenko, KA;Wanner, BL
通讯作者：
Wanner, BL