X-ray structure of 5-aminolaevulinate dehydratase, a hybrid aldolase

Abstract

5-Aminolaevulinate dehydratase (ALAD) is a homo-octameric metallo-enzyme that catalyses the formation of porphobilinogen from 5-aminolaevulinic acid. The structure of the yeast enzyme has been solved to 2.3 A resolution, revealing that each subunit adopts a TIM barrel fold with a 39 residue N-terminal arm. Pairs of monomers wrap their arms around each other to form compact dimers and these associate to form a 422 symmetric octamer. All eight active sites are on the surface of the octamer and possess two lysine residues (210 and 263), one of which, Lys 263, forms a Schiff base link to the substrate. The two lysine side chains are close to two zinc binding sites one of which is formed by three cysteine residues (133, 135 and 143) while the other involves Cys 234 and His 142. ALAD has features at its active site that are common to both metallo- and Schiff base-aldolases and therefore represents an intriguing combination of both classes of enzyme. Lead ions, which inhibit ALAD potently, replace the zinc bound to the enzyme's unique triple-cysteine site.

Mechanistic Implications of Mutations to the Active Site Lysine of Porphobilinogen Synthase

“…Equilibrium Dialysis to Determine the Active Site Stoichiometry for E. coli PBGS-A stoichiometry of four active sites per octamer is consistent with previous biochemical studies of bovine, human, E. coli, Bradyrhizobium japonicum, and Pseudomonas aeruginosa PBGS (2, 4, 12, 13, 15, 26 -29) and is seen in the crystal structure of P. aeruginosa PBGS (8), but is not obvious from the crystal structures of yeast or E. coli PBGS (6,7). We propose that any PBGS can exist as either a symmetric or asymmetric octamer depending upon the presence or absence of certain active site ligands, as has been shown for human PBGS with regard to Zn(II) binding (4).…”

“…However, since to date 13 C-labeled porphobilinogen bound to all Zn(II)-requiring PBGS has shown identical chemical shifts (2,12), 13 C-labeled porphobilinogen bound to K252G is not expected to provide different information than that available from the E. coli PBGS variant K246G. In the case of K246W, only free [3,[5][6][7][8][9][10][11][12][13] C]porphobilinogen (and a degradation product of unknown structure) was observed at both pH 7 and pH 8. The line widths of free [3,[5][6][7][8][9][10][11][12][13] C]porphobilinogen and the degradation product were quite sharp (ϳ7 Hz) confirming their unbound state.…”

“…By analogy to wild type, we assume that the more upfield signal (127-129 ppm range) arises from C-5 and the more downfield signal (121-124 ppm range) arises from C-3. In all cases the C-5 signal of enzyme-bound product is 6.3-7.7 ppm upfield from the chemical shift of free [3,[5][6][7][8][9][10][11][12][13] C]porphobilinogen. From this we conclude that the active site lysine is not a major contributor to the massive shift seen at C-5.…”

“…For wild-type E. coli PBGS, the control samples, which included all the physical manipulations to the protein, retained complete catalytic activity. In contrast, when treated with both [4][5][6][7][8][9][10][11][12][13][14] C]ALA and NaBH 4 , three samples of wild-type PBGS each showed ϳ80% inactivation and 14 C labeling at 0.42-0.49 ALA/subunit. This stoichiometry is consistent with four functional active sites per octamer.…”

Crystal Structure of Toxoplasma gondii Porphobilinogen Synthase

“…Statistics of data collection parameters are summarized in Table 1. TgPBGS crystal structure determination follows that of yeast, Escherichia coli, Pseudomonas aeruginosa, Chlorobium vibrioforme, human, and mouse PBGS proteins, which are all homo-oligomeric enzymes, with a common subunit structure minimally consisting of an ␣␤-barrel domain and an N-terminal arm domain (22). In all known PBGS proteins, the N-terminal arm is essential for oligomer assembly.…”