Lee Y S, Cho Y D
Department of Biochemistry, College of Science, Yonsei University, Seoul 120-749, Korea.
Biochem J. 2001 Dec 15;360(Pt 3):657-65. doi: 10.1042/0264-6021:3600657.
The cDNA encoding ornithine decarboxylase (ODC; EC 4.1.1.17), a key enzyme in putrescine and polyamine biosynthesis, has been cloned from Nicotiana glutinosa (GenBank AF 323910), and was expressed in Escherichia coli. The amino acid sequence of N. glutinosa ODC showed 90% identity with Datura stramonium ODC, and 44% identity with human ODC. N. glutinosa ODC did not possess the PEST sequence [a sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues] found in mammalian ODCs, which are thought to be involved in rapid degradation of the protein. The purified ODC was a homodimeric protein, having a native M(r) of 92000. Kinetic studies of ODC showed that N. glutinosa ODC decarboxylated both l-ornithine and l-lysine with K(m) values of 562 microM and 1592 microM at different optimal pH values of 8.0 and 6.8 respectively. ODC activity was completely and irreversibly inhibited by alpha-difluoromethylornithine (K(i) 1.15 microM), showing a competitive inhibition pattern. Site-directed mutagenesis was performed on ODC to introduce mutations at conserved lysine (Lys(95)) and cysteine (Cys(96), Cys(338) and Cys(377)) residues, chosen by examination of the conserved sequence, which were proven by chemical modification to be involved in enzymic activity. Except for Cys(96), each mutation caused a substantial loss in enzyme activity. Most notably, Lys(95) increased the K(m) for l-ornithine by 16-fold and for l-lysine by 3-fold, with 100-fold and 2.8-fold decreases in the k(cat) for ODC and lysine decarboxylase (LDC) activity respectively. The Cys(377)-->Ala mutant possessed a k(cat) that was lowered by 23-fold, and the K(m) value was decreased by 1.4-fold for l-ornithine. The three-dimensional model of ODC protein constructed on the basis of the crystal structure of Trypanosoma brucei, mouse and human ODCs localized the four residues in the active-site cleft. This is the first work carried out on active-site residues of plant ODC, where ODC and LDC activities occur in the same catalytic site.
编码鸟氨酸脱羧酶（ODC；EC 4.1.1.17）的cDNA已从粘毛烟草（GenBank AF 323910）中克隆出来，该酶是腐胺和多胺生物合成中的关键酶，并在大肠杆菌中表达。粘毛烟草ODC的氨基酸序列与曼陀罗ODC有90%的同一性，与人类ODC有44%的同一性。粘毛烟草ODC不具有在哺乳动物ODC中发现的PEST序列[富含脯氨酸（P）、谷氨酸（E）、丝氨酸（S）和苏氨酸（T）残基的序列]，据认为该序列与蛋白质的快速降解有关。纯化后的ODC是一种同二聚体蛋白，天然相对分子质量为92000。ODC的动力学研究表明，粘毛烟草ODC在不同的最佳pH值（分别为8.0和6.8）下，对L-鸟氨酸和L-赖氨酸进行脱羧反应，其米氏常数（K(m)）分别为562 microM和1592 microM。ODC活性被α-二氟甲基鸟氨酸完全且不可逆地抑制（抑制常数K(i)为1.15 microM），呈现竞争性抑制模式。对ODC进行定点诱变，在保守的赖氨酸（Lys(95)）和半胱氨酸（Cys(96)、Cys(338)和Cys(377)）残基处引入突变，这些残基是通过检查保守序列选择的，经化学修饰证明它们参与酶活性。除了Cys(96)外，每个突变都导致酶活性大幅丧失。最显著的是，Lys(95)使L-鸟氨酸的K(m)增加了16倍，使L-赖氨酸的K(m)增加了3倍，ODC和赖氨酸脱羧酶（LDC）活性的催化常数（k(cat)）分别降低了100倍和2.8倍。Cys(377)→Ala突变体的k(cat)降低了23倍，L-鸟氨酸的K(m)值降低了1.4倍。基于布氏锥虫、小鼠和人类ODC的晶体结构构建的ODC蛋白三维模型将这四个残基定位在活性位点裂隙中。这是首次对植物ODC的活性位点残基进行的研究，其中ODC和LDC活性发生在同一个催化位点。
