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Identification of residues in ornithine decarboxylase essential for enzymic activity and for rapid protein turnover.

ABSTRACT: The importance of certain amino acid residues in mammalian ornithine decarboxylase activity and degradation was studied by site-specific mutagenesis. Changes were made to the mouse ornithine decarboxylase cDNA in a plasmid containing a T7 RNA polymerase promoter. The plasmid was then used for the synthesis of RNA, which was translated in a reticulocyte lysate system. The activity of the ornithine decarboxylase formed and the stability of the protein to degradation in a reticulocyte lysate system were determined. Changes of lysine-169 or of histidine-197 to alanine completely abolished enzyme activity, indicating that these residues are essential for enzyme activity. The removal of the C-terminal 36 residues, the mutation of lysine-349 to alanine, of lysine-298 to alanine or the double change of serine-303 and glutamic acid-308 to alanine residues still resulted in an active enzyme. The last-mentioned finding indicates that the phosphorylation of serine-303 does not play an essential role in the catalytic activity of ornithine decarboxylase. The control ornithine decarboxylase protein was degraded rapidly in a reticulocyte lysate provided that ATP was added. The truncated protein missing the 36 residues from the C-terminus was much more stable in this system, and the protein containing the double change of serine-303 and glutamic acid-308 to alanine residues was slightly more stable than control ornithine decarboxylase protein. These results indicate that the altered residues may play a role in interaction with factors responsible for the rapid turnover of ornithine decarboxylase.

SUBMITTER: Lu L

PROVIDER: S-EPMC1151295 | biostudies-other | 1991 Aug

REPOSITORIES: biostudies-other

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Project description: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.

Project description:The prostate is a male accessory sex gland that produces secretions in seminal fluid to facilitate fertilization. Prostate secretory function is dependent on androgens, although the mechanism by which androgens exert their effects is still unclear. Polyamines are small cationic molecules that play pivotal roles in DNA transcription, translation and gene regulation. The rate-limiting enzyme in polyamine biosynthesis is ornithine decarboxylase, which is encoded by the gene Odc1. Ornithine decarboxylase mRNA decreases in the prostate upon castration and increases upon administration of androgens. Furthermore, testosterone administered to castrated male mice restores prostate secretory activity, whereas administering testosterone and the ornithine decarboxylase inhibitor D,L-α-difluromethylornithine (DFMO) to castrated males does not restore prostate secretory activity, suggesting that polyamines are required for androgens to exert their effects. To date, no one has examined polyamines in prostate development, which is also androgen dependent. In this study, we showed that ornithine decarboxylase protein was expressed in the epithelium of the ventral, dorsolateral and anterior lobes of the adult mouse prostate. Ornithine decarboxylase protein was also expressed in the urogenital sinus (UGS) epithelium of the male and female embryo prior to prostate development, and expression continued in prostatic epithelial buds as they emerged from the UGS. Inhibiting ornithine decarboxylase using DFMO in UGS organ culture blocked the induction of prostatic buds by androgens, and significantly decreased expression of key prostate transcription factor, Nkx3.1, by androgens. DFMO also significantly decreased the expression of developmental regulatory gene Notch1. Other genes implicated in prostatic development including Sox9, Wif1 and Srd5a2 were unaffected by DFMO. Together these results indicate that Odc1 and polyamines are required for androgens to exert their effect in mediating prostatic bud induction, and are required for the expression of a subset of prostatic developmental regulatory genes including Notch1 and Nkx3.1.

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Identification of residues in ornithine decarboxylase essential for enzymic activity and for rapid protein turnover.

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