van den Bosch M, Swart M, van Gunsteren W F, Canters Gerard W
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.
J Mol Biol. 2004 Nov 26;344(3):725-38. doi: 10.1016/j.jmb.2004.09.056.
Molecular dynamics (MD) simulations have been performed on quercetin 2,3 dioxygenase (2,3QD) to study the mobility and flexibility of the substrate cavity. 2,3QD is the only firmly established Cu-containing dioxygenase known so far. It catalyses the breakage of the O-heterocycle of flavonols. The substrates occupy a shallow and overall hydrophobic cavity proximal to the metal centre of the homo-dimeric enzyme. The linker connecting the C-terminal and N-terminal domains in the monomer is partly disordered in the crystal structure and part of it forms a flexible lid at the entrance of the substrate cavity. This loop has been tentatively assigned a role in the enzyme mechanism: it helps lock the substrate into place. The dynamics of this loop has been investigated by MD simulation. The initial coordinates were taken from the crystal structure of 2,3QD in the presence of the substrate kaempferol (KMP). After equilibration and simulation over 7.2ns the substrate was removed and another equilibration and simulation of 7.2ns was performed. The results show that the structures of the free enzyme as well as of the enzyme-substrate complex are stable in MD simulation. The linker shows strongly enhanced mobility in the loop region that is close to the entrance to the substrate cavity (residues 154-169). Movement of the loop takes place on a timescale of 5-10ns. To confirm the conclusions about the loop dynamics drawn from the 7.2ns simulation, the simulation was extended with another 8ns. When substrate binds into the cavity the loop orders remarkably, although mobility is retained by residues 155-158. Some regions of the loop (residues 154-160 and 164-176) move over a considerable distance and approach the substrate closely, reinforcing the idea that they lock the substrate in the substrate cavity. The enthalpic component of the interaction of the loop with the protein and the KMP appears to favour the locking of the substrate. Two water molecules were found immobilised in the cavity, one of which exhibited rotation on the picosecond timescale. When the substrate is removed, the empty cavity fills up with water within 200ps.
已对槲皮素2,3 -双加氧酶（2,3QD）进行了分子动力学（MD）模拟，以研究底物腔的流动性和灵活性。2,3QD是目前已知的唯一一种确定含铜的双加氧酶。它催化黄酮醇的O -杂环断裂。底物占据同二聚体酶金属中心附近的一个浅且总体疏水的腔。单体中连接C末端和N末端结构域的连接子在晶体结构中部分无序，其一部分在底物腔入口处形成一个灵活的盖子。这个环已被初步认为在酶机制中起作用：它有助于将底物锁定到位。已通过MD模拟研究了这个环的动力学。初始坐标取自2,3QD与底物山奈酚（KMP）结合时的晶体结构。在平衡和7.2纳秒的模拟后，移除底物并进行了另一次7.2纳秒的平衡和模拟。结果表明，在MD模拟中，游离酶以及酶 - 底物复合物的结构是稳定的。连接子在靠近底物腔入口的环区域（残基154 - 169）显示出显著增强的流动性。环的运动发生在5 - 10纳秒的时间尺度上。为了证实从7.2纳秒模拟得出的关于环动力学的结论，模拟又延长了8纳秒。当底物结合到腔中时，环显著有序，尽管残基155 - 158保留了流动性。环的一些区域（残基154 - 160和164 - 176）移动了相当大的距离并靠近底物，强化了它们将底物锁定在底物腔中的观点。环与蛋白质和KMP相互作用的焓成分似乎有利于底物的锁定。发现有两个水分子固定在腔中，其中一个在皮秒时间尺度上表现出旋转。当底物被移除时，空的腔在200皮秒内充满水。
