X-ray analysis of several enzymes involved in the biosynthesis of tetrapyrroles such as haem and chlorophyll is underway in collaboration with Professor Peter Jordan (Southampton). Genetic deficiencies in these enzymes give rise to various hereditory porphyrias.
We have determined the structure of porphobilinogen deaminase (PBGD), the enzyme which catalyses the stepwise polymerisation of four molecules of its pyrrole substrate, porphobilinogen, yielding a linear tetrapyrrole which is then cyclised and rearranged by subsequent enzymes in the pathway.
Our analysis of E. coli PBGD at 1.8 Angstrom resolution shows the enzyme to consist of three domains (labelled 1,2 and 3) with the third domain binding the enzyme's dipyrromethane cofactor via a covalent link to Cys 242. During catalysis, the cofactor acts as an anchor on which the growing polypyrrole chain is assembled within the enzyme's cleft. Domains 1 and 2, which bear a strong topological resemblence to other anion binding proteins, form the active site cleft of the enzyme and contain a large number of invariant basic residues which interact with the pyrrole carboxyl groups.
The above figure shows the tertiary structure of porphobilinogen deaminase with the dipyrromethane cofactor occupying the cleft between domains 1 and 2.
Site-directed mutagenesis indicates that catalysis probably stems from a single aspartate group (residue 84) which is positioned close to the cofactor's pyrrole nitrogens. X-ray analyses of site-directed mutants are underway to determine the mechanism by which each incoming pyrrole can reach the active site during elongation. Whether the assembling polypyrrole folds into a cavity next to the cofactor or whether the growing chain is pulled through the cleft by movement of domain 3 remains to be established.
We are also working on the enzyme 5-aminolaevulinate dehydratase which catalyses the synthesis of porphobilinogen from two 5-aminolaevulinate (ALA) residues. This octameric zinc-dependent enzyme has no significant homology with other protein families and is notably sensitive to inhibition by lead ions. The crystals diffract to 2.0 Angstrom resolution at 100 Kelvin and analysis of the structure by multiple isomorphous replacement is in progress.
Researchers: N.Picken, P.Erskine, R.Lambert, J.Cooper, S.P.Wood & T.L.Blundell.
Key reference.
The three dimensional structure of porphobilinogen deaminase: a flexible multidomain polymerase with one catalytic site.
G.V.Louie, P.D.Brownlie, R.Lambert, J.B.Cooper, T.L.Blundell, S.P.Wood, M.J.Warren, S.C.Woodcock and P.M.Jordan. Nature (1992) 359, 33-39.