Caldinelli Laura, Molla Gianluca, Pilone Mirella S, Pollegioni Loredano
Department of Biotechnology and Molecular Sciences, University of Insubria, Varese, Italy.
FEBS J. 2006 Feb;273(3):504-12. doi: 10.1111/j.1742-4658.2005.05083.x.
The flavoenzyme d-amino acid oxidase from Rhodotorula gracilis is a homodimeric protein whose dimeric state has been proposed to occur as a result of (a) the electrostatic interactions between positively charged residues of the betaF5-betaF6 loop of one monomer and negatively charged residues belonging to the alpha-helices I3' and I3'' of the other monomer, and (b) the interaction of residues (e.g. Trp243) belonging to the two monomers at the mixed interface region. The role of Trp243 was investigated by substituting it with either tyrosine or isoleucine: both substitutions were nondisruptive, as confirmed by the absence of significant changes in catalytic activity, but altered the tertiary structure (yielding a looser conformation) and decreased the stability towards temperature and denaturants. The change in conformation interferes both with the interaction of the coenzyme to the apoprotein moiety (although the kinetics of the apoprotein-FAD complex reconstitution process are similar between wild-type and mutant D-amino acid oxidases) and with the interaction between monomers. Our results indicate that, in the folded holoenzyme, Trp243 is situated at a position optimal for increasing the interactions between monomers by maximizing van der Waals interactions and by efficiently excluding solvent.
来自纤细红酵母的黄素酶D-氨基酸氧化酶是一种同二聚体蛋白，其二聚体状态被认为是由于：(a) 一个单体的βF5-βF6环带正电荷残基与另一个单体的α-螺旋I3'和I3''带负电荷残基之间的静电相互作用；(b) 两个单体在混合界面区域的残基（如Trp243）之间的相互作用。通过用酪氨酸或异亮氨酸取代Trp243来研究其作用：两种取代均无破坏性，这通过催化活性无显著变化得到证实，但改变了三级结构（产生更松散的构象）并降低了对温度和变性剂的稳定性。构象变化既干扰辅酶与脱辅基蛋白部分的相互作用（尽管野生型和突变型D-氨基酸氧化酶之间脱辅基蛋白-FAD复合物重组过程的动力学相似），也干扰单体之间的相互作用。我们的结果表明，在折叠的全酶中，Trp243位于一个最佳位置，通过最大化范德华相互作用和有效排除溶剂来增加单体之间的相互作用。
