Chung Jung-Sung, Noguera-Mazon Valérie, Lancelin Jean-Marc, Kim Sung-Kun, Hirasawa Masakazu, Hologne Maggy, Leustek Thomas, Knaff David B
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061, USA.
J Biol Chem. 2009 Nov 6;284(45):31181-9. doi: 10.1074/jbc.M109.035634. Epub 2009 Sep 10.
NMR spectroscopy has been used to map the interaction domain on Escherichia coli thioredoxin for the thioredoxin- dependent 5'-adenylylsulfate reductase from Pseudomonas aeruginosa (PaAPR). Seventeen thioredoxin amino acids, all clustered around Cys-32 (the more surface-exposed of the two active-site cysteines), have been located at the PaAPR binding site. The center of the binding domain is dominated by nonpolar amino acids, with a smaller number of charged and polar amino acids located on the periphery of the site. Twelve of the amino acids detected by NMR have non-polar, hydrophobic side chains, including one aromatic amino acid (Trp-31). Four of the thioredoxin amino acids at the PaAPR binding site have polar side chains (Lys-36, Asp-61, Gln-62 and Arg-73), with three of the four having charged side chains. Site-directed mutagenesis experiments have shown that replacement of Lys-36, Asp-61, and Arg-73 and of the absolutely conserved Trp-31 significantly decreases the V(max) for the PaAPR-catalyzed reduction of 5'-adenylylsulfate, with E. coli thioredoxin serving as the electron donor. The most dramatic effect was observed with the W31A variant, which showed no activity as a donor to PaAPR. Although the thiol of the active-site Cys-256 of PaAPR is the point of the initial nucleophilic attack by reduced thioredoxin, mutagenic replacement of Cys-256 by serine has no effect on thioredoxin binding to PaAPR.
核磁共振光谱已被用于绘制大肠杆菌硫氧还蛋白上与铜绿假单胞菌硫氧还蛋白依赖性5'-腺苷硫酸还原酶（PaAPR）相互作用的结构域。十七个硫氧还蛋白氨基酸都聚集在Cys-32（两个活性位点半胱氨酸中更暴露于表面的那个）周围，已定位在PaAPR结合位点。结合结构域的中心以非极性氨基酸为主，在该位点的外围有较少数量的带电荷和极性氨基酸。通过核磁共振检测到的氨基酸中有十二个具有非极性、疏水侧链，包括一个芳香族氨基酸（Trp-31）。在PaAPR结合位点的四个硫氧还蛋白氨基酸具有极性侧链（Lys-36、Asp-61、Gln-62和Arg-73），其中四个中的三个具有带电荷的侧链。定点诱变实验表明，用大肠杆菌硫氧还蛋白作为电子供体时，替换Lys-36、Asp-61和Arg-73以及绝对保守的Trp-31会显著降低PaAPR催化的5'-腺苷硫酸还原反应的V(max)。观察到W31A变体的影响最为显著，它作为PaAPR的供体没有活性。尽管PaAPR活性位点Cys-256的硫醇是被还原的硫氧还蛋白最初亲核攻击的位点，但用丝氨酸诱变替换Cys-256对硫氧还蛋白与PaAPR的结合没有影响。
