The Principle of the Redox Activity Expression for FMN-binding Protein Based on its Higher Structure

采用蛋白质工程方法对硫酸盐还原菌中fmn结合蛋白（FMN-bp）进行了研究。FMN-bp的氨基酸序列与黄伏氧蛋白的氨基酸序列相比较，黄伏氧蛋白与FMN具有几乎相同的分子量，并结合FMN作为一个假基，只有3 - trp - asn序列是相同的。采用定点诱变技术，通过基因工程产生了9个FMN-bps突变体，讨论了该序列对FMN结合的影响，并以藏红花红T为介质测定了FMN的氧化还原电位。将Trp残基转化为Ala、Tyr、Phe或His残基的突变体的氧化还原电位均为正（+8 ~ +18 mV）。这一结果表明，色氨酸残基可能与氧化还原电位无关。然而，这一残基被证明对结合FMN很重要。另一方面，将Asn残基转变为Gin或Asp残基的突变体在结合FMN和氧化还原电位方面差异不大。Asn残基在Thr-Trp-Asn中最不保守，结果表明Asn残基在这些残基中最不重要。将Thr残基转化为Ser残基或Val残基的突变体的氧化还原电位分别为负（5mv）或负（约60mv）。这一结果表明，该位置保持亲水性对氧化还原电位有重要作用。然后对Thr残基变为Val残基的突变体进行结晶，并用x射线测定其高级结构。我们获得了分辨率超过1.5A的数据，现在我们提出了它的模型。

I studied FMN-binding protein (FMN-bp) from the sulfate-reducing bacteria by use of protein-engineering methods. Comparing the amino acid sequence of FMN-bp with that of flavodoxin, which has almost same molecular weight and binds FMN as a prosthetic group, the only Thir-Trp-Asn sequence is the same each other. Using site-directed mutagenesis, 9 mutant FMN-bps were produced by genetic engineering, and were disucussed about the influences of this sequence on the binding of FMN and the redox potential which was measured using safranine T as a mediator. The redox potential of every mutant that changed Trp residue to Ala, Tyr, Phe or His residue shifted rather positive (+8 to +18 mV). This result suggested that Trp residue might not be responsible on redox potential. However, this residue was indicated to be important on binding FMN. On the other hand, the mutant that changed Asn residue to Gin or Asp residue showed little difference on both binding FMN and redox potential. The Asn residue is least conserved in Thr-Trp-Asn and my result indicated that Asn residue was least important in these residues. The redox potential of the mutant that changed Thr residue to Ser or Val residue shifted rather (5 mV) or much (about 60 mV) negative, respectively. This result indicated that it is important on redox potential that this position keeps hydrophilic. Then the mutant that changed Thr residue to Val residue was crystallized and determined the higher structure using x-ray. We obtained the data whose resolution was over 1.5A and now we are proposing its model.