| Ornithine decarboxylase: Expression and regulation in rat brain and in transgenic mice | ||
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All eukaryotic ODCs contain several highly conserved regions and the amino acid residues 232-238 form one of the most highly conserved sequences. We mutated aspartete-233 which is the only acidic residue in the -LD(I/V)GGGF- motif to valine. The mutation resulted in increases in the Km values for the substrate L-ornithine and the cofactor PLP by about 20-fold. Ki value for the DFMO increased 15-fold. Glycine-rich sequences like this have been implicated in the formation of the cofactor binding region in the PLP-dependent enzymes (Marceau et al. 1988a, Marceau et al. 1988b).
According to the crystal structure of mouse ODC (Kern et al. 1999), the position and orientation of the PLP in the active site is governed primarily by protein interaction with the 5’-phosphate and the pyrimidine nitrogen, N1. The phosphate binds in a pocket formed by two loops (235-241, 274 – 276) and the aminoterminus of 10’th helix that follows immediately the second loop. The first loop contains three consecutive glycines (235 – 237) that are conserved in all group IV PLP-dependent decarboxylases (Kern et al. 1999). Glycine-237 and –276, arginine-277 and tyrosine-389 form hydrogen bonds to the phosphate of PLP. The primary interaction of N1 is with glutamate-274. Glutamate-274 forms together with aspartate-88, glutamate-274 and aspartate-233 a cluster of acidic residues that, with three bound water molecules form a network of hydrogen bonds that probably influences the electron-withdrawing properties of the cofactor. It is understandable that the mutation of aspartate-233 can effect PLP-binding, or the orientation or position of the cofactor and thus also the substrate binding.
Other glycine-rich regions which interact with phosphate groups of nucleotides are found in the guanine nucleotide binding domains. The PM1 region in c-H-ras protein (de Vos et al. 1988, Valencia et al. 1991) has similarities in amino acid sequence with mammalian ODC, especially if conservative amino acid substitutions are allowed. The most apparent of similarities is between amino acids 230 – 240 of ODC and 5 – 15 of c- H-ras. The amino acid sequence of residues 230 - 240 in ODC is -HLLDIGGGØØG- and that of residues 5 - 15 of c-H-ras is KLVVVGAGØØG-. Homologous, either identical or similar, amino acids are written in italics, amino acids designated Ø are not thought to be indispensable for GTP-binding (Möller & Amons 1985, de Vos et al. 1988, Valencia et al. 1991). However, the aspartate-233 to valine-233 mutation did not, at least alone, cause the activation of ODC by GTP.