Porphobilinogen synthase (PBGS) catalyzes the first common step in the biosynthesis of tetrapyrroles (such as heme and chlorophyll). Although the predominant oligomeric form of this enzyme, as inferred from many crystal structures, is that of a homo-octamer, a rare human PBGS allele, F12L, reveals the presence of a hexameric form. Rearrangement of an N-terminal arm is responsible for this oligomeric switch, which results in profound changes in kinetic behavior. The structural transition between octamer and hexamer must proceed through an unparalleled equilibrium containing two different dimer structures. The allosteric magnesium, present in most PBGS, has a binding site in the octamer but not in the hexamer. The unprecedented structural rearrangement reported here relates to the allosteric regulation of PBGS and suggests that alternative PBGS oligomers may function in a magnesium-dependent regulation of tetrapyrrole biosynthesis in plants and some bacteria.

The enzyme porphobilinogen synthase (PBGS) can exist in different non-additive homo-oligomeric assemblies and, under appropriate conditions, the distribution of these assemblies can respond to ligands such as metals or substrate. PBGS from most organisms was believed to be octameric until work on a rare allele of human PBGS revealed an alternate hexameric assembly, which is also available to the wild type enzyme at elevated pH. Herein, we establish that the distribution of pea PBGS quaternary structures also contains octamers and hexamers, using both sedimentation velocity and sedimentation equilibrium experiments. We report results in which the octamer dominates under purification conditions and discuss conditions that influence the octamer:hexamer ratio. As predicted by PBGS crystal structures from related organisms, in the absence of magnesium, the octameric assembly is significantly destabilized and the oligomeric distribution is dominated largely by the hexameric assembly. Although the PBGS hexamer-to-octamer oligomeric rearrangement is well document under some conditions, both assemblies are very stable (under AUC conditions) in the timeframe of our ultracentrifuge experiments.The enzyme porphobilinogen synthase (PBGS), also known as 5-aminolevulinic acid dehydratase (ALAD), catalyzes the first common step in the biosynthesis of tetrapyrroles including heme, chlorophyll, vitamin B 12 and cofactor F 430 (1,2). The pea (Pisum sativum) PBGS enzyme has been a useful model system to explore metal ion regulation of enzyme activity (2). It has been proposed that part of the regulatory mechanism for pea PBGS activity involves dynamic control of its oligomeric assembly (3) and Mg 2+ has been implicated in the quaternary structure of PBGS from species such as Escherichia coli, Pseudomonas aeruginosa, and Chlorobium vibrioforme (4-6).The dynamic nature of the quaternary structure of pea PBGS under assay conditions results in a protein concentration dependent specific enzyme activity (2). This unusual kinetic phenomenon was originally interpreted as due to an additive equilibrium of oligomeric assemblies that could include less active dimers and tetramers along with active octamers (2); the octamer was well established from the first crystal structure of PBGS (7). A more recent structural hypothesis ascribes the low activity of pea PBGS to a hexameric state, which is nonadditive relative to the octamer, and analogous to the recently determined crystal structure of † This work was supported by NSF grants MCB-0211754 and MCB-0510625 to R.F. and NIH grants ES003654 (EKJ), AI063324 (EKJ), a hexameric form of human PBGS (3). The interconversion of the human PBGS octamer and hexamer is now well established to proceed via a mechanism of dissociation to a dimeric assembly, conformational change at the level of the dimer, and reassociation to the alternate higher order oligomer (8,9). The interpretation of pea PBGS as assembling to either an octamer or a hexamer, as illustrated in Figure 1, is supported by structural a...

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