Domain organization and a protease-sensitive loop in eukaryotic ornithine decarboxylase.
Trypanosoma brucei ornithine decarboxylase was reconstituted by coexpression of two polypeptides corresponding to residues 1-305 and residues 306-425 in Escherichia coli. The two peptides were coexpressed, at wild-type levels, from a single transcriptional unit that was separated by a 15-nucleotide untranslated region containing a ribosome binding site. The fragmented enzyme was purified and analyzed. The N- and C-terminal peptides are tightly associated into a fully active tetramer which has the same molecular weight as the native dimer. The kinetic constants (Km and kcat) measured for the decarboxylation of ornithine are identical to those obtained for the wild-type enzyme. These results suggest that the enzyme is organized into two structural domains, with a domain boundary in the region of amino acid 305. In contrast, the individual N- and C-terminal peptides are expressed primarily as inclusion bodies. Small quantities of soluble N-terminal peptide could be purified. This truncated protein is capable of inhibiting the wild-type enzyme, suggesting that it is folded into a native-like structure. Limited proteolysis with trypsin or chymotrypsin identifies a likely surface loop at amino acids 160-170, present in both the mouse and T. brucei enzyme, which positions one or more functionally important active site residues (e.g., Lys169). Kinetic analysis of a chimeric enzyme composed of T. brucei and mouse ornithine decarboxylase suggests that the substrate carboxylate binding determinant is located between residues 1 and 170.
Osterman
A L
AL
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235, USA.
Lueder
D V
DV
Quick
M
M
Myers
D
D
Canagarajah
B J
BJ
Phillips
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002918:Chymotrypsin / Q000378:metabolism*; D004926:Escherichia coli; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009955:Ornithine Decarboxylase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000201:enzymology*; D014357:Trypsin / Q000378:metabolism*
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D002918:Chymotrypsin; D014357:Trypsin; D009955:Ornithine Decarboxylase