【作者】 陈辉；

【导师】 滕脉坤； 牛立文；

【作者基本信息】 中国科学技术大学 ， 结构生物学， 2009， 博士

【摘要】 (1) 5-磷酸吡哆醛依赖酶是一类以5-磷酸吡哆醛为辅基的酶类。这类酶家族蛋白在自然界分布非常广泛,种类繁多,催化的反应种类多样,其功能是参与氨基酸和氨基酸衍生物的生物合成和降解途径。根据此类蛋白空间结构,将其分为五类折叠类型,分别为转氨酶家族（第一类折叠）,色氨酸合成酶家族（第二类折叠）,丙氨酸消旋酶家族（第三类折叠）,D-氨基酸转氨酶家族（第四类）和肝糖磷酸酶家族（第五类折叠）。转氨酶蛋白家族能可逆的催化生物体中a-氨基酸和酮酸之间的氨基的转移作用,这对于氨基酸的代谢至关重要。底物特异性和序列特性决定了转氨酶的四个亚类。我们研究的酿酒酵母ARO9蛋白属于转氨酶中的第一个亚类。酿酒酵母ARO9蛋白可逆性催化芳香族氨基酸的转氨反应。我们克隆了该蛋白的基因,在大肠杆菌中得到过量表达,在纯化和结晶该蛋白后,根据分子置换法解析了该蛋白的2.4埃的晶体结构。酶活证明底物为苯丙氨酸时的比活力为8.21U/mg,酶活动力学分析得到K_m值为0.85mmol l^-1、V_m值为2.87mmol l^-1 min^-1、K_cat值为11.6min^-1。ARO9的结构包括三个部分:位于N-端的区域,大结构域和小结构域。其中大结构域和小结构域存在于所有转氨酶家族中。ARO9的大结构域含有7个反平行的β折叠片和16个a螺旋。位于C-端的小结构域折叠成3个反平行的β折叠片和5个a螺旋。结构比对结果说明ARO9结构的最大不同之处在于N-端区域。ARO9的N-端序列有2个反平行的β折叠片。这2个反平行的β折叠片和ARO9同二体的形成有关。序列比对和结构比对证实ARO9和人源犬尿酸转氨酶Ⅱ9（hKatⅡ）组成了转氨酶第Ⅰ家族中的一个新亚类,而且关键的活性氨基酸残基的空间构象与hKatⅡ一致,推测ARO9的催化机制与hKatⅡ类似,N端序列（32-40）控制酶活中心的“开”或“关”。（2）人源ZO-2蛋白是一个含有多个结构域的蛋白,属于MAGUK家族。这些结构域包括一个SH3结构域,一个GUK结构域和3个PDZ结构域。这3个PDZ结构域调节蛋白蛋白之间的相互作用,从而影响蛋白蛋白之间的聚集以及蛋白在细胞中的定位。我们使用分子置换法获得了人源ZO-2的第二个结构域1.75埃分辨率的晶体结构。晶体结构表明ZO-2 PDZ2能形成稳定的同二体结构。二体结构的形成通过链交换的方式形成。结构比对和docking实验说明ZO-2PDZ2的同二体结构是ZO-2 PDZ2与Connexin43蛋白的C端序列小肽结合的前提,而且小肽结合的方式与典型PDZ结合方式不同。更多还原

【Abstract】 The PLP-dependent enzymes are the enzymes which use pyridoxal-5’-phosphate as cofactor.They are widely distributed in nature,and catalyze a large variety of reactions.These enzymes are principally participated in the catabolism and the anabolism of amino acids and amino acid-derived metabolites.Based on the solved structure,all the PLP enzymes are divided into five fold types:the fold typeⅠ(aspartate aminotransferase family),the fold typeⅡ(tryptophan synthase family),the fold typesⅢ(alanine racemase family),the fold typeⅣ(D-amino acid aminotransferase family) and the fold typeⅤ(glycogen phosphorylase family).The aminotransferases catalyze the reversible transfer of amino groups from amino acids to oxo acids.This is vital for the metabolism of amino acids.Based on the substrate specificity and the sequence specificity,these aminotransferases are divided into four subfamilies.Saccharomyces cerevisae ARO9 protein,an aromatic aminotransferaseⅡ,belongs to the aminotransferase subfamilyⅠ.It catalyzes the transamination step of the catabolism of aromatic amino acids.We reported the crystal structure of ARO9,determined by molecular replacement method at 2.4(?).The crystal structure of ARO9 consists by three regions:the N-terminal region, the large domain and the small domain.The large and small domains are conserved among the aspirated aminotransferase.The large domain contains a seven-strandedβ-sheet and 16 helices.And the small domain comprises the C-terminal part,which folds into three-strandedβ-sheet and 5 helices.Three dimensional structure comparisons revealed that the major difference in protein folding to be at the N-terminal residues.In ARO9,there are two antiparallelβstrands.The region is critical in forming the homodimer of ARO9.Alignments of the sequence and structure of ARO9 with other aminotransferase indicated that ARO9 and hKatⅡactually form a new subgroup in subfamilyⅠaminotransferase.It also indicated that the residues in the catalytic sites were lacated at the same spatial positions as hKatⅡ. (2) Human ZO-2 protein is a multi-domain protein consisting of an SH3 domain, a GUK domain and three copies of PDZ domain with slightly divergence.The three PDZ domains act as protein-recognition modules that may mediate protein assembly and subunit localization.The crystal structure of the second PDZ domain of ZO-2 (ZO-2 PDZ2) was determined by molecular replacement,revealing a dimer per asymmetric unit at 1.75(?) resolution.The dimer is stabilized by extensive symmetrical domain-swapping ofβ1 andβ2 strands.Structural comparison and docking show that ZO-2 PDZ2 homodimer might have a difference ligand-binding pattern to the classic PDZ binding pattern.更多还原