Abstract:
: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.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Osterman AL,Lueder DV,Quick M,Myers D,Canagarajah BJ,Phillips MAdoi
10.1021/bi00041a021subject
Has Abstractpub_date
1995-10-17 00:00:00pages
13431-6issue
41eissn
0006-2960issn
1520-4995journal_volume
34pub_type
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