Mutational analysis confirms the presence of distal inhibitor-selectivity determining residues in B. stearothermophilus dihydrofolate reductase.
Mutational analysis confirms the presence of distal inhibitor-selectivity determining residues in B. stearothermophilus dihydrofolate reductase.
复制标题
突变分析证实嗜热脂肪芽孢杆菌二氢叶酸还原酶中存在远端抑制剂选择性决定残基。
DOI：
10.1016/j.abb.2020.108545
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发表时间：
2020
影响因子：
3.9
通讯作者：
Goodey,NinaM
中科院分区：
文献类型：
--
作者：
Eck,Tyler;Patel,Seema;Candela,Thomas;LeonH,Katherine;Little,Michael;Reis,NataliaE;Liyanagunawardana,Uththara;Gubler,Ueli;Janson,CherylA;Catalano,Jaclyn;Goodey,NinaM
许多抗菌和抗寄生虫药物通过竞争性抑制二氢叶酸还原酶（DHFR）发挥作用，DHFR是叶酸代谢中的一种重要酶。抑制剂和DHFR活性位点残基之间的相互作用在许多同系物中是已知的，但远端残基的贡献还不太清楚。鉴定有助于抑制剂结合的远端残基可以通过解释可能不太保守且易受导致突变的频繁耐药的远端影响来改善靶向药物开发计划。以前，一种新的，同源性为基础的，计算的方法，地雷配体抑制数据被用来预测残基参与抑制剂的选择性DHFR家族。预期的是，一些抑制剂选择性决定残基位置被预测位于活性位点中，并且与实验已知的抑制剂选择性决定位置一致。然而，其他的残基，组在空间上的集群远端的活性位点还没有以前的调查。在这项研究中，在这些预测的集群之一（His38-Ala39-Ile40）的抑制剂的选择性概况在嗜热脂肪芽孢杆菌二氢叶酸还原酶（BsDHFR）的氨基酸取代的影响进行了研究。将突变引入这些簇位置以改变侧链化学和大小。测定了两种竞争性DHFR抑制剂甲氧苄啶（TMP）和乙胺嘧啶（PYR）的kcat和Km值，并测量了平衡时的KD值。His38-Ala39-Ile40簇中的突变显著影响抑制剂结合和TMP/PYR选择性-当与TMP相比时，9个突变中的7个导致与PYR更紧密的结合。这些数据表明，His38-Ala39-Ile40簇是一个远端抑制剂选择性决定区，有利于PYR结合BsDHFR，并可能，在整个DHFR家族。
Many antibacterial and antiparasitic drugs work by competitively inhibiting dihydrofolate reductase (DHFR), a vital enzyme in folate metabolism. The interactions between inhibitors and DHFR active site residues are known in many homologs but the contributions from distal residues are less understood. Identifying distal residues that aid in inhibitor binding can improve targeted drug development programs by accounting for distant influences that may be less conserved and subject to frequent resistance causing mutations. Previously, a novel, homology-based, computational approach that mines ligand inhibition data was used to predict residues involved in inhibitor selectivity in the DHFR family. Expectedly, some inhibitor selectivity determining residue positions were predicted to lie in the active site and coincide with experimentally known inhibitor selectivity determining positions. However, other residues that group spatially in clusters distal to the active site have not been previously investigated. In this study, the effect of introducing amino acid substitutions at one of these predicted clusters (His38-Ala39-Ile40) on the inhibitor selectivity profile inBacillus stearothermophilusdihydrofolate reductase (BsDHFR) was investigated. Mutations were introduced into these cluster positions to change sidechain chemistry and size. We determinedkcatand KMvalues and measured KDvalues at equilibrium for two competitive DHFR inhibitors, trimethoprim (TMP) and pyrimethamine (PYR). Mutations in the His38-Ala39-Ile40 cluster significantly impacted inhibitor binding and TMP/PYR selectivity - seven out of nine mutations resulted in tighter binding to PYR when compared to TMP. These data suggest that the His38-Ala39-Ile40 cluster is a distal inhibitor selectivity determining region that favors PYR binding inBsDHFR and, possibly, throughout the DHFR family.