【作者】 何永兴；

【导师】 周丛照；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 2,4-二乙酰基间苯酚(DAPG)是由荧光假单胞杆菌产生的一种可以有效杀伤细菌、真菌和线虫的抗生素,在高剂量下甚至对植物也有杀伤作用。PhlG作为一种碳-碳键水解酶,参与DAPG代谢的过程。它可以水解与间苯酚相连的一个乙酰基,把高毒性的DAPG,转变成低毒性的单乙酰基间苯酚(MAPG)。我们用多波长反常散射法(MAD)解析了PhlG的晶体结构,分辨率达到2.0 A。晶体结构显示,PhlG以二体形式存在,单体是由较小的一个N端二体化结构域和一个C端的所谓的Bet vl-like fold组成。PhlG的这种折叠类型,有别于以往发现的经典的α/β-fold碳-碳键水解酶的结构。通过结构分析,我们发现,在PhlG内部,有一个狭小的口袋,同时分布着疏水残基和极性残基,以及一个五配位的锌离子。突变及活性分析显示,与锌离子配位的残基突变,会导致PhlG的kcat/Km下降约5个数量级,提示与锌络合的水分子很可能在水解过程中参与了亲核攻击的作用。通过将底物DAPG与PhlG的结构进行分子对接,我们确定了参与稳定底物的残基Tyr121、Tyr229和Asn132。进一步通过突变和活性分析,我们确证了我们计算获得的酶-底物复合物模型的合理性,而以上这三个氨基酸残基在反应过程中参与稳定和结合底物。最后,我们还发现,PhlG中存在一条从活性中心指向溶剂的狭长的通道。底物DAPG进入活性中心将引起排布于该通道的残基发生较大的构象变化。对位于该通道的Cys的谷胱甘肽化的修饰以及活性分析进一步确证了这个底物进入通道的合理性。细胞色素b5是一类含有血红素的电子传递蛋白,广泛的存在于动植物、真菌和蓝细菌中。它们参与脂肪酸的去饱和、胆固醇的合成和细胞色素P450依赖的氧化和羟化反应。我们解析了酿酒酵母的细胞色素b5(Cyb5)和它的还原酶Cbrl的晶体结构。值得注意的是,细胞色素b5的在晶体结构中呈二体结构,其中的一个单体的血红素分子指向另一个分子的α3螺旋,此外,Asp46和Asp42的侧链分布在血红素分子的丙酸基的两边,其中Asp46直接与血红素分子的丙酸基形成氢键,而这两个残基与细胞色素b5和细胞色素c的相互作用有关。因此,我们观察到的Cyb5二聚体的界面,很可能对应与细胞色素b5与其相互作用蛋白的作用界面。我们获得了Cyb5与Cbr1的复合物的模型,希望能够加深对细胞色素b5的电子传递过程的了解。谷氨酰胺合酶(GS, EC 6.3.1.2)在氮元素的同化中起着关键的作用。它通过水解ATP,催化谷氨酸和氨形成谷氨酰胺。目前已经有玉米、人和犬类的谷氨酰胺合酶的晶体结构相继被报道。我们解析了酿酒酵母的谷氨酰胺合酶Glnl的晶体结构,分辨率为2.95A。在谷氨酸结合位点有一个柠檬酸分子,通过结构比对发现,柠檬酸分子结合使得Leu293-Ala300这一段loop的柔性变大,因此我们推测,这一段loop很可能负责控制谷氨酸进入活性中心。此外,Glnl的晶体结构呈现二十聚体,除了已经报道的十聚体的作用界面外,还有一种背靠背的五元环的相互作用,这个界面可以使Glnl装配成纳米管状的超分子结构,但对于它的生物学意义,目前还尚不清楚。更多还原

【Abstract】 2,4-Diacetylphloroglucinol hydrolase PhlG from Pseudomonas fluorescens catalyzes hydrolytic carbon-carbon (C-C) bond cleavage of the antibiotic 2,4-diacetylphloroglucinol (DAPG) to form monoacetylphloroglucinol (MAPG), a rare class of reaction in chemistry and biochemistry. To investigate the catalytic mechanism of this enzyme, we determined the three-dimensional structure of PhlG at 2.0 (?) resolution using X-ray crystallography and multi-wavelength anomalous dispersion (MAD) methods. The overall structure comprises a small N-terminal domain mainly involved in dimerization and a C-terminal domain of Bet vl-like fold, which distinguishes PhlG from the classicalα/β-fold hydrolases. A dumbbell shaped substrate access tunnel was identified to connect a narrow interior amphiphilic pocket to the exterior solvent. The tunnel is likely to undergo a significant conformational change upon substrate binding to the active site. Structural analysis coupled with computational docking studies, site-directed mutagenesis, and enzymatic activity analysis revealed that cleavage of the DAPG C-C bond proceeds via nucleophilic attack by a water molecule, which is coordinated by a zinc ion. In addition, residues Tyr121, Tyr229 and Asn132, which are predicted to be hydrogen-bonded to the hydroxyl groups and unhydrolyzed acetyl group, can finely tune and position the bound substrate in a reactive orientation. Taken together, these results revealed the active sites and zinc-dependent hydrolytic mechanism of PhlG and explained its substrate specificity as well.Cytochromes b5 are electron transfer hemoproteins ubiquitous in animals, plants, fungi and purple phototrophic bacteria. They are involved in a broad spectrum of reactions, including fatty acid desaturation, cholesterol biosynthesis, and cytochrome P450-dependent oxidation and hydroxylation reactions. Here, we report the crystal structures of cytochrome b5 and its reductase from the yeast Saccharomyces cerevisiae. Notably, Cyb5 monomers were assembled into dimers in the crystalline environment and the solvent-exposed edge of the haem group faces the interface between the two monomers, with one of the propionate groups pointing towards the helixα3 of the neighboring subunit. Also, the side-chains of Asp46 and Asp42, shown to be critical for electron transfer from cytochrome b5 to cytochrome c, sandwiched the propionate group of pyrrole II with Asp46 forming a direct hydrogen bond with it. Using computational docking method, we constructed the complex model of Cyb5 and Cbr1, which could shed light on the structural basis of the electron transfer between Cyb5 and Cbrl.Glutamine synthetase (GS, EC 6.3.1.2) plays an essential role in nitrogen assimilation by catalyzing the condensation of glutamate and ammonium to form glutamine, with concomitant hydrolysis of ATP. Following the recent publication of maize, human and canine GSII structures, we report the crystal structure of Glnl from the yeast Saccharomyces cerevisiae at the resolution of 2.95 A, with a citrate molecule binding to each glutamate binding site. Comparative structure analysis suggests that citrate binding could induce structural fluctuation of the segment Leu293-Ala300, which may serve the role of guarding the glutamate entrance to the active site. Besides a decameric quaternary structure and active sites similar to the published GSII structures, a novel back-to-back inter-ring interface was found. This additional interface enables Glnl to be assembled into a nanotube-like supramolecule, although the biological significance is still an open question.更多还原