Structural and Mechanistic Studies of 10-Formyltetrahydrofolate Synthetase

甲酰基四氢叶酸合成酶（FTHFS）利用ATP水解的能量来催化四氢叶酸的可逆甲酰化反应。所有已知的fthfs都是同源的；然而，在许多真核生物中，fthfs是三功能c -1-四氢叶酸合酶的一部分，而在细菌中它是一个单独的酶。FTHFS的机制已经研究了近三十年，但由于缺乏有关其三维结构的信息，进展有限。特别令人感兴趣的是单价阳离子在催化中的作用，叶酸结合位点与其他酶中存在的位点的比较。感兴趣的酶来自嗜热细菌，热稳定性的结构来源将被分析。本研究是基于以前对热醋酸梭菌10-甲酰基四氢叶酸合成酶的研究。早期的工作确定了dna和氨基酸序列，在大肠杆菌中表达了该酶，纯化并结晶了该酶的重组天然形式和硒代蛋氨酸形式。从Se-Met晶体中采集了2.5 A分辨率的多波长异常色散（MAD）数据。用直接法确定了异常散射体的位置，并将结果用于解决相位问题。建立了该分子的模型，并用晶体学方法对其进行了优化。进一步的研究将指向确定酶与底物/催化中间体和抑制剂络合物形式的结构。根据结构数据，将阐明活性物中存在的侧链的催化作用。这样产生的假设将使用定点诱变进行测试。

Lukasz LebiodaMCB 987360610-Formyltetrahydrofolate synthetase (FTHFS) catalyzes the reversibleformylation of tetrahydrofolate using the energy of ATP hydrolysis to drivethe reaction. All known FTHFSs are homologous; however, in many eukaryotesFTHFS is a part of trifunctional C-1-tetrahydrofolate synthase while inbacteria it is a separate enzyme. The mechanism of FTHFS has been studiedfor almost three decades but in the absence of information about itsthree-dimensional structure the progress has been limited. Of particularinterest is the role of monovalent cations in catalysis, the comparison ofthe folate-binding site with those present in other enzymes. The enzyme ofinterest is from thermophilic bacteria and the structural origins ofthermostability will be analyzed.This study is based on previous studies with 10-Formyltetrahydrofolatesynthetase from Clostridium thermoaceticum. Earlier work determined thecDNA and amino acid sequence, expressed the enzyme in E. coli, purified andcrystallized the recombinant native and selenomethionine forms of theenzyme. Multi-wavelength anomalous dispersion (MAD) data were collected to2.5 A resolution from the Se-Met crystals. Positions of anomalousscatterers were determined using direct methods and the results used tosolve the phase problem. A model of the molecule was built and optimizedusing crystallographic refinement. Further studies will be directed towardsthe determination of the structures of the enzyme in the form of complexeswith the substrates/catalytic intermediates, and inhibitors. Based onstructural data, the catalytic role of side chains present in the activesite will be elucidated. The hypotheses so generated will be tested usingsite-directed mutagenesis.