The cDNA encoding ornithine decarboxylase (ODC; EC 184.108.40.206), 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 Mr of 92000. Kinetic studies of ODC showed that N. glutinosa ODC decarboxylated both l-ornithine and l-lysine with Km values of 562μM and 1592μM at different optimal pH values of 8.0 and 6.8 respectively. ODC activity was completely and irreversibly inhibited by α-difluoromethylornithine (Ki 1.15μM), showing a competitive inhibition pattern. Site-directed mutagenesis was performed on ODC to introduce mutations at conserved lysine (Lys95) and cysteine (Cys96, Cys338 and Cys377) residues, chosen by examination of the conserved sequence, which were proven by chemical modification to be involved in enzymic activity. Except for Cys96, each mutation caused a substantial loss in enzyme activity. Most notably, Lys95 increased the Km for l-ornithine by 16-fold and for l-lysine by 3-fold, with 100-fold and 2.8-fold decreases in the kcat for ODC and lysine decarboxylase (LDC) activity respectively. The Cys377 → Ala mutant possessed a kcat that was lowered by 23-fold, and the Km 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.
Identification of essential active-site residues in ornithine decarboxylase of Nicotiana glutinosa decarboxylating both l-ornithine and l-lysine
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Yong-Sun LEE, Young-Dong CHO; Identification of essential active-site residues in ornithine decarboxylase of Nicotiana glutinosa decarboxylating both l-ornithine and l-lysine. Biochem J 15 December 2001; 360 (3): 657–665. doi: https://doi.org/10.1042/bj3600657
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