Identification of essential active-site residues in ornithine decarboxylase of Nicotiana glutinosa decarboxylating both l-ornithine and l-lysine

Abstract

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 (GenBank2 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 92 000. Kinetic studies of ODC showed that N. glutinosa ODC decarboxylated both -ornithine and -lysine with K m values of 562 µM and 1592 µM at different optimal pH values of 8.0 and 6.8 respectively. ODC activity was completely and irreversibly inhibited by α-difluoromethylornithine (K i 1.15 µM), showing a competitive inhibition pattern. Site-directed mutagenesis was performed on ODC to introduce mutations at conserved lysine Abbreviations used : ADC, arginine decarboxylase ; C377A, mutant bearing the site-directed mutation of Cys 377 Ala, etc. ; DFMO, α-

Immunomodulation of polyamine biosynthesis in tobacco plants has a significant impact on polyamine levels and generates a dwarf phenotype

“…Before studying the in vivo effect of selected scFv fragments on polyamine metabolism in tobacco plants, the cross‐reactivity between scFvODC1, 3, 7 and 15 recognizing the human ODC and the endogenous tobacco ODC enzyme was analysed by immunoblot analysis. scFvODC1 detected a single protein of approximately 47 kDa in total soluble protein (TSP) extracts from wild‐type tobacco plants, corresponding to the known molecular mass of ODC from Nicotiana glutinosa and other plants (Lee and Cho, 2001) (Figure 1a). In contrast, no endogenous proteins were detected by scFvODC3 (Figure 1b), scFvODC7 and scFvODC15 (data not shown) or the anti‐c‐myc antibody which was chosen as the negative control (Figure 1c).…”

“…The ability of scFvODC1 to bind both human and tobacco ODC proteins indicates that this recombinant antibody fragment recognizes a highly conserved epitope on the eukaryotic ODC enzyme. The active site of eukaryotic ODC is highly conserved, and previous studies have indicated that the mutation of amino acid residues localized at the active site cleft is critical for ODC activity (Coleman et al ., 1993; Michael et al ., 1996; Lee and Cho, 2001). Therefore, we speculate that the epitope recognized by scFvODC1 is located within or close to the active site of ODC.…”

“…Considering that eukaryotic ODC is active as a homodimeric molecule, whereas the monomer retains no enzymatic activity (Tobias et al ., 1993), binding of recombinant antibody fragment could induce conformational changes of the monomeric ODC preventing the formation of the active homodimeric ODC enzyme. Site‐directed mutagenesis of an evolutionarily conserved glycine residue, which plays an essential role in dimer formation, completely abolishes ODC enzymatic activity (Tobias et al ., 1993; Lee and Cho, 2001).…”

The scaffold-forming steps of plant alkaloid biosynthesis

“…Ornithine decarboxylation is the typical route in eukaryotes. 43 Ornithine decarboxylases (OrnDCs) have been identied and characterised from nicotine 45 and tropane alkaloid 46,47 producing plants. An alternative route to putrescine proceeds via arginine.…”

Polyamines in Microalgae: Something Borrowed, Something New

“…In general, ODC from the mouse, humans, plants, and some parasites is known to have a very strong preference for ornithine as a substrate [76,77,78]. However, ODC may also have weak enzymatic activity toward Lys and Arg [79,80]. Moreover, there is a unique ODC homolog (PBCV1 DC) present in the genome of the Paramecium Bursaria Chlorella virus, which shows a preference for Arg as a substrate.…”