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 structural basis for allosteric regulation of phenylalanine hydroxylase (PAH), whose dysfunction causes phenylketonuria (PKU), is poorly understood. A new morpheein model for PAH allostery is proposed to consist of a dissociative equilibrium between two architecturally different tetramers whose interconversion requires a ~90° rotation between the PAH catalytic and regulatory domains, the latter of which contains an ACT domain. This unprecedented model is supported by in vitro data on purified full length rat and human PAH. The conformational change is both predicted to and shown to render the tetramers chromatographically separable using ion exchange methods. One novel aspect of the activated tetramer model is an allosteric phenylalanine binding site at the inter-subunit interface of ACT domains. Amino acid ligand-stabilized ACT domain dimerization follows the multimerization and ligand binding behavior of ACT domains present in other proteins in the PDB. Spectroscopic, chromatographic, and electrophoretic methods demonstrate a PAH equilibrium consisting of two architecturally distinct tetramers as well as dimers. We postulate that PKU-associated mutations may shift the PAH quaternary structure equilibrium in favor of the low activity assemblies. Pharmacological chaperones that stabilize the ACT:ACT interface can potentially provide PKU patients with a novel small molecule therapeutic.

Improved understanding of the relationship among structure, dynamics, and function for the enzyme phenylalanine hydroxylase (PAH) can lead to needed new therapies for phenylketonuria, the most common inborn error of amino acid metabolism. PAH is a multidomain homo-multimeric protein whose conformation and multimerization properties respond to allosteric activation by the substrate phenylalanine (Phe); the allosteric regulation is necessary to maintain Phe below neurotoxic levels. A recently introduced model for allosteric regulation of PAH involves major domain motions and architecturally distinct PAH tetramers [Jaffe EK, Stith L, Lawrence SH, Andrake M, Dunbrack RL, Jr (2013) Arch Biochem Biophys 530(2):73-82]. Herein, we present, to our knowledge, the first X-ray crystal structure for a full-length mammalian (rat) PAH in an autoinhibited conformation. Chromatographic isolation of a monodisperse tetrameric PAH, in the absence of Phe, facilitated determination of the 2.9 Å crystal structure. The structure of full-length PAH supersedes a composite homology model that had been used extensively to rationalize phenylketonuria genotype-phenotype relationships. Small-angle X-ray scattering (SAXS) confirms that this tetramer, which dominates in the absence of Phe, is different from a Phestabilized allosterically activated PAH tetramer. The lack of structural detail for activated PAH remains a barrier to complete understanding of phenylketonuria genotype-phenotype relationships. Nevertheless, the use of SAXS and X-ray crystallography together to inspect PAH structure provides, to our knowledge, the first complete view of the enzyme in a tetrameric form that was not possible with prior partial crystal structures, and facilitates interpretation of a wealth of biochemical and structural data that was hitherto impossible to evaluate.phenylalanine hydroxylase | phenylketonuria | X-ray crystallography | small-angle X-ray scattering | allosteric regulation M ammalian phenylalanine hydroxylase (PAH) (EC 1.14.16.1) is a multidomain homo-multimeric protein whose dysfunction causes the most common inborn error in amino acid metabolism, phenylketonuria (PKU), and milder forms of hyperphenylalaninemia (OMIM 261600) (1). PAH catalyzes the hydroxylation of phenylalanine (Phe) to tyrosine, using nonheme iron and the cosubstrates tetrahydrobiopterin and molecular oxygen (2, 3). A detailed kinetic mechanism has recently been derived from elegant single-turnover studies (4). PAH activity must be carefully regulated, because although Phe is an essential amino acid, high Phe levels are neurotoxic. Thus, Phe allosterically activates PAH by binding to a regulatory domain. Phosphorylation at Ser16 potentiates the effects of Phe, with phosphorylated PAH achieving full activation at lower Phe concentrations than the unphosphorylated protein (5, 6). Allosteric activation by Phe is accompanied by a major conformational change, as evidenced by changes in protein fluorescence and proteolytic susceptibility, and by stabilization of a tetrameric confo...

(2003) Nat. Struct. Biol. 10, 757-763). The quaternary structure isoforms of PBGS result from two alternative conformations of the monomer; one monomer structure assembles into a high activity octamer, whereas the other monomer structure assembles into a low activity hexamer. The kinetic behavior of these oligomers led to the hypothesis that turnover facilitates the interconversion of the oligomeric structures. The current work demonstrates that the interactions of ligands at the enzyme active site promote the structural interconversion between human PBGS quaternary structure isoforms, favoring formation of the octamer. This observation illustrates that the assembly and disassembly of oligomeric proteins can be facilitated by the protein motions that accompany enzymatic catalysis.A recent description of a rare human allele of porphobilinogen synthase (PBGS) 1 revealed that the enzyme can exist as two alternate quaternary structures wherein the oligomeric state is dictated by distinctly different structures for their component crystallographic asymmetric unit (1). The monomer is an ␣␤-barrel protein with a 23-residue N-terminal arm. The octameric form is comprised of four "hugging dimers" (Fig. 1a), whereas the hexameric form is comprised of three "detached dimers" (Fig. 1b). In both oligomeric assemblies there are invariant barrel-to-barrel subunit interactions within the dimers and quasiequivalent arm-to-bottom-of-barrel subunit interactions between adjacent dimers (Fig. 1). However, the octamer has a unique "hugging" arm-to-barrel interaction that is not seen in the hexamer.Preparations of heterologously expressed wild type human PBGS predominantly contain octamer, but there is a small propensity to adopt the hexameric structure (1). In contrast, heterologous expression of the rare human variant, F12L, yields protein that purifies as the hexamer and does not readily take on the octameric structure. The homo-octameric wild type and homohexameric F12L forms of human PBGS exhibit dramatic differences in pH activity profile and in the kinetic K m values (Table I) (1) despite the fact that amino acid residue 12 does not interact directly with active site residues in either quaternary structure isoform (Fig. 1). However, the arm-tobarrel interface found in the hugging dimer provides substantial stabilization for the solvent-accessible face of the ␣␤-barrel wherein lies the active site. This region of the protein, which is disordered in the hexamer, includes an ϳ11-amino acid stretch that normally serves to gate the active site from solvent, also called the active site lid. With the active site more solventaccessible in the hexamer, a high external pH is required to drive the Schiff base formation that is necessary for catalysis (2); this contributes to the high pH required for F12L activity. The decreased affinity of F12L for substrate is attributed to the loss of interactions between the destabilized active site lid and the carboxyl moiety of the outermost substrate (3).Coexpression of wild type and F12L (WTϩF12L...

Evidence is building to support the notion that the porphobilinogen synthase (PBGS 2 ; EC 4.2.1.24) family of enzymes can exist as an equilibrium of quaternary structure isoforms, denoted morpheeins (1-3). Morpheeins comprise an equilibrium ensemble of protein structures wherein a protein monomer can exist in more than one conformation, and each monomer conformation dictates a functionally different quaternary structure of finite multiplicity. Morpheeins have been proposed to provide a structural foundation for allosteric regulation, cooperativity, and hysteresis in some proteins (2). As such, the energetic difference between morpheeins of a given protein must be small. The propensity of PBGS to assume various morpheein structures and the rates of PBGS morpheein interconversion are highly species-dependent. The stable morpheeins of human PBGS are the octamer, found for the wild-type protein, and the hexamer, first seen for the naturally occurring mutation F12L (1). Coexpression of human wild-type PBGS and F12L generates a population of PBGS proteins composed of hetero-octamers and heterohexamers, each of which contains a mixture of Phe 12 -and Leu 12 -containing chains (1). The structure and composition of these hetero-oligomers are stable during storage, but the molecular motions resulting from catalysis favor formation of the octamer with an accompanying disproportionation of Phe 12 -containing chains into the octamer (3).The remaining hexamer has an increased proportion of Leu 12 -containing chains (3). The physical basis for the thermodynamic propensity of Leu 12 -containing chains to form the hexamer remains unclear, but examination of the structure of human PBGS suggests that other single amino acid mutations might affect the folding and assembly of the protein to favor structures other than the octamer. In this study, we report on alterations of two amino acids (Arg 240 and Trp 19 ) that were chosen based on an analysis of the structures and subunit interactions seen in human octameric and hexameric PBGS, for which the assemblies are shown in Fig. 1. Each human PBGS subunit is composed of a 306-amino acid TIM-like ␣␤-barrel and a 24-amino acid N-terminal arm. The conformational difference between the monomer that assembles into the octamer and the monomer that assembles into the hexamer is a dramatic reorientation of the arm with respect to the barrel; for the F12L hexamer, this reorientation occurs at Thr 23 . In both the octameric and hexameric assemblies, two monomers come together to form a dimer with a conserved barrel-barrel interface. The dimer that assembles into the octamer is called a hugging dimer (Fig. 1a), whereas the dimer that assembles into the hexamer is called a detached dimer (Fig. 1b) (1). The difference between these two dimers is the presence or absence of a "hugging" interaction between the N-terminal arm of one subunit and the ␣␤-barrel of the adjacent subunit of the dimer. The "arm-hugging-barrel" interaction of PBGSs from plants, Archaea, and most Bacteria is stabilized by an all...

ALAD porphyria is a rare porphyric disorder, with five documented compound heterozygous patients, and it is caused by a profound lack of porphobilinogen synthase (PBGS) activity. PBGS, also called "delta-aminolevulinate dehydratase," is encoded by the ALAD gene and catalyzes the second step in the biosynthesis of heme. ALAD porphyria is a recessive disorder; there are two common variant ALAD alleles, which encode K59 and N59, and eight known porphyria-associated ALAD mutations, which encode F12L, E89K, C132R, G133R, V153M, R240W, A274T, and V275M. Human PBGS exists as an equilibrium of functionally distinct quaternary structure assemblies, known as "morpheeins," in which one functional homo-oligomer can dissociate, change conformation, and reassociate into a different oligomer. In the case of human PBGS, the two assemblies are a high-activity octamer and a low-activity hexamer. The current study quantifies the morpheein forms of human PBGS for the common and porphyria-associated variants. Heterologous expression in Escherichia coli, followed by separation of the octameric and hexameric assemblies on an ion-exchange column, showed that the percentage of hexamer for F12L (100%), R240W (80%), G133R (48%), C132R (36%), E89K (31%), and A274T (14%) was appreciably larger than for the wild-type proteins K59 and N59 (0% and 3%, respectively). All eight porphyria-associated variants, including V153M and V275M, showed an increased propensity to form the hexamer, according to a kinetic analysis. Thus, all porphyria-associated human PBGS variants are found to shift the morpheein equilibrium for PBGS toward the less active hexamer. We propose that the disequilibrium of morpheein assemblies broadens the definition of conformational diseases beyond the prion disorders and that ALAD porphyria is the first example of a morpheein-based conformational disease.

Porphobilinogen synthase (PBGS) catalyzes the first common step in tetrapyrrole (e.g. heme, chlorophyll) biosynthesis. Human PBGS exists as an equilibrium of high activity octamers, low activity hexamers, and alternate dimer configurations that dictate the stoichiometry and architecture of further assembly. It is posited that small molecules can be found that inhibit human PBGS activity by stabilizing the hexamer. Such molecules, if present in the environment, could potentiate disease states associated with reduced PBGS activity, such as lead poisoning and ALAD porphyria, the latter of which is associated with human PBGS variants whose quaternary structure equilibrium is shifted toward the hexamer (Jaffe, E. K., and Stith, L. (2007) Am. J. Hum. Genet. 80, 329 -337). Hexamer-stabilizing inhibitors of human PBGS were identified using in silico prescreening (docking) of ϳ111,000 structures to a hexamer-specific surface cavity of a human PBGS crystal structure. Seventyseven compounds were evaluated in vitro; three provided 90 -100% conversion of octamer to hexamer in a native PAGE mobility shift assay. Based on chemical purity, two (ML-3A9 and ML-3H2) were subjected to further evaluation of their effect on the quaternary structure equilibrium and enzymatic activity. Naturally occurring ALAD porphyria-associated human PBGS variants are shown to have an increased susceptibility to inhibition by both ML-3A9 and ML-3H2. ML-3H2 is a structural analog of amebicidal drugs, which have porphyria-like side effects. Data support the hypothesis that human PBGS hexamer stabilization may explain these side effects. The current work identifies allosteric ligands of human PBGS and, thus, identifies human PBGS as a medically relevant allosteric enzyme.Human porphobilinogen synthase (PBGS, 2 EC 4.2.1.24, also known as 5-aminolevulinate dehydratase) exists as a quaternary structure equilibrium consisting of a high activity octamer, a low activity hexamer, and a dimer that can take on two distinct conformations, each of which dictates assembly to either the octamer or the hexamer (see Fig. 1a) (1). For the wild type protein at neutral pH, the octamer is the dominant assembly in the equilibrium (1). As PBGS catalyzes the first common step in the biosynthesis of the tetrapyrroles such as heme, low activity mutations in the human population are associated with the disease ALAD porphyria (2); all eight porphyria-associated PBGS mutations increase the propensity of the human protein to exist in the low activity hexameric assembly, thus establishing the physiologic relevance of the quaternary structure equilibrium to human health (3). In addition to ALAD porphyria, PBGS inhibition by divalent lead is a primary consequence of lead poisoning. Factors that stabilize the hexamer will further inhibit PBGS activity and, thus, potentiate the physiologic effects of lead poisoning.The arrangement of the subunits in the PBGS hexamer creates a surface cavity that is not present in the octamer or the dimers (see Fig. 1b) (4). Ligand binding to this cavi...

Porphobilinogen synthase (PBGS) proteins fall into several distinct groups with different metal ion requirements. Drosophila melanogaster porphobilinogen synthase (DmPBGS) is the first non-mammalian metazoan PBGS to be characterized. The sequence shows the determinants for two zinc binding sites known to be present in both mammalian and yeast PBGS, proteins that differ in the exhibition of half-of-the-sites metal binding. The pH-dependent activity of DmPBGS is uniquely affected by zinc. A tight binding catalytic zinc binds at 0.5/subunit with a K d well below M. A second inhibitory zinc exhibits a K d of ϳ5 M and appears to bind at a stoichiometry of 1/subunit. A molecular model of DmP-BGS suggests that the inhibitory zinc is located at a subunit interface using Cys-219 and His-10 as ligands. Zinc binding to this previously unknown inhibitory site is proposed to inhibit opening of the active site lid. As predicted, the DmPBGS mutant H10F is active but is not inhibited by zinc. H10F binds a catalytic zinc at 0.5/ subunit and binds a second nonessential and noninhibitory zinc at 0.5/subunit. This result reveals a structural basis for half-of-the-sites metal binding that is consistent with a reciprocating motion model for function of oligomeric PBGS. The porphobilinogen synthases (PBGS)1 are a family of highly homologous homo-octameric proteins responsible for catalyzing the first common step in the biosynthesis of a broad range of tetrapyrrole pigments such as heme, vitamin B12, chlorophyll, and cofactor F430 of the methanogenic bacteria (1). PBGS is also known as 5-aminolevulinate dehydratase or ALAD. The most significant phylogenetic difference among PBGS proteins is in the constellation of metal ions at catalytic and allosteric sites (2, 3). Yeast and mammalian PBGS share the sequence determinants for two zinc binding sites, one of which is absent from the PBGS of any archaea, bacteria, protist, or photosynthetic eucarya (3). The human and yeast PBGS both contain the sequence determinants for a catalytic active site zinc (also known as ZnB), as well as a second non-essential zinc (also known as ZnA) (4), but they differ in metal binding stoichiometry. For instance, in the case of human PBGS, the catalytic zinc shows half-of-the-sites binding at a stoichiometry of 4/homo-octamer (4) (PDB accession number 1E51), 2 whereas the fungal enzyme binds the catalytic zinc at 8/octamer (6) (PDB accession number 1AW5). Some bacterial PBGS show half-of-the-sites metal binding at catalytic and/or allosteric sites (e.g. Bradyrhizobium japonicum and Pseudomonas aeruginosa), whereas others (e.g. Escherichia coli) do not.The current sequence databases contain PBGS sequences from ϳ130 different organisms. Of these, the PBGS of Drosophila melanogaster (DmPBGS) is the only complete non-mammalian and non-fungal PBGS sequence that shows the sequence determinants for the two zinc binding sites found in yeast and mammalian PBGS. The gene encoding PBGS is absent from the completed genome of Caenorhabditis elegans and not yet verified in...