X-ray Structure, Sequence and Solution Properties of Ornithine Decarboxylase from Lactobacillus 30a

Summary

The polyamines, putrescine, spermidine and spermine, are essential for all cells and their concentrations within cells are highly regulated. The synthesis of polyamines begins with the conversion of ornithine into putrescine which is also the starting material for the synthesis of spermidine and spermine. In mammalian cells, fungi, and most protozoa the only route available to synthesize putrescine is via the enzyme ornithine decarboxylase, a pyridoxal phosphate-dependent enzyme. We have determined the gene sequence and X-ray structure of ornithine decarboxylase (ODC) from Lactobacillus 30a. This ODC is a dodecamer of ~1MDa that crystallizes in space group P6, a = b = 195.6Å, c = 97.6Å with two 730 a.a. residue monomers/ asymmetric unit.

The structure of the PLP-dependent decarboxylase is markedly different from what we found for pyruvoyl-dependent histidine decarboxylase. In the latter case the functional unit is a trimer of αβ subunits, while in the PLP-dependent decarboxylases the functional unit is a dimer. Each dimer consists of a tightly packed “core” involving monomer/monomer contacts plus protruding “wing” domains that interlock with adjacent dimers to form the ring-like dodecamer. Each monomer may be further broken down into five folding domains: the N-terminal 107 residues form the 5-stranded β-sheet “wing” domain, residues 108–161 form a linker domain, residues 162–415 make up the PLP-binding scaffold which is reminiscent of the 7-stranded β-sheet observed in aspartate aminotransferase, and the final two domains (residues 416–571 and 572–730) help create a cleft that leads to the active site at the subunit interface deep within the dimeric “core.”

Using the ODC structure as a guide, we have identified amino acid sequence motifs common to decarboxylases, transaminases, and other PLP-dependent enzymes. In particular, the conserved residues Asp316, A1a318, and Lys355 of ODC assume identical functional roles as in the aminotransferases. We have also identified a GTP effector site which has the effect of broadening the pH range over which the enzyme is active. The GTP site lies on the surface of the protein at the interface between the two subunits, but approximately 27Å from the PLP binding site. Kinetic studies show that the effector maintains enzyme activity by shifting the sharp rise in Km from pH 7 to pH 9, but that Vmax remains nearly constant over the pH range of 4 to 9. Preparation of inhibitor complexes and site-directed mutants to explore the functional role of individual residues are in progress.