Biochemical and molecular characterization of a novel porphobilinogen synthase from Corynebacterium glutamicum.

Corynebacterium glutamicum porphobilinogen synthase (PBGS) is a metal enzyme with a hybrid active site metal binding sequence. In this study, the porphobilinogen synthase gene of C. glutamicum was cloned and heterogeneously expressed in Escherichia coli. C. glutamicum PBGS was purified, and its enzymatic characteristics were analyzed. The results showed that C. glutamicum PBGS is a Zn2+-dependent enzyme, and Mg2+ has allosteric regulation. The allosteric Mg2+ plays a vital role in forming the quaternary structure of C. glutamicum PBGS. Based on the ab initio predictive structure modeling of the enzyme and the molecular docking model of 5-aminolevulinic acid (5-ALA), 11 sites were selected for site-directed mutagenesis. When the hybrid active site metal binding site of C. glutamicum PBGS is converted into a cysteine-rich motif (Zn2+-dependent) or an aspartic acid-rich motif (Mg2+/K+-dependent), the enzyme activity is basically lost. Four residues, D128, C130, D132, and C140, in the metal binding site, were the binding sites of Zn2+ and the active center of the enzyme. The band migration, from the native PAGE, of five variants with mutations in the center of enzyme activity was the same as that of the variant enzymes as purified, individually adding two metal ion chelating agents. Their Zn2+ active center structures were abnormal, and the quaternary structure equilibrium was altered. The destroyed active center affects the construction of its quaternary structure. The quaternary structural balance between octamer and hexamer through dimers was regulated by the allosteric regulation of C. glutamicum PBGS. The enzyme activity was also affected by the change of the active site lid structure and (α ß)8-barrel structure caused by mutation. Structural changes in the variants were analyzed to understand C. glutamicum PBGS better.

Exploring human porphobilinogen synthase metalloprotein by quantum biochemistry and evolutionary methods.

Previous studies have shown the porphobilinogen synthase (PBGS) zinc-binding mechanism and its conservation among the living cells. However, the precise molecular interaction of zinc with the active center of the enzyme is unknown. In particular, quantum chemistry techniques within the density functional theory (DFT) framework have been the key methodology to describe metalloproteins, when one is looking for a compromise between accuracy and computational feasibility. Considering this, we used DFT-based models within the molecular fractionation with conjugate caps scheme to evaluate the binding energy features of zinc interacting with the human PBGS. Besides, phylogenetic and clustering analyses were successfully employed in extracting useful information from protein sequences to identify groups of conserved residues that build the ions-binding site. Our results also report a conservative assessment of the relevant amino acids, as well as the benchmark analysis of the calculation models used. The most relevant intermolecular interactions in Zn2+-PBGS are due to the amino acids CYS0122, CYS0124, CYS0132, ASP0169, SER0168, ARG0221, HIS0131, ASP0120, GLY0133, VAL0121, ARG0209, and ARG0174. Among these residues, we highlighted ASP0120, GLY0133, HIS0131, SER0168, and ARG0209 by co-occurring in all clusters generated by unsupervised clustering analysis. On the other hand, the triple cysteines at 2.5 Å from zinc (CYS0122, CYS0124, and CYS0132) have the highest energy attraction and are absent in the taxa Viridiplantae, Sar, Rhodophyta, and some Bacteria. Additionally, the performance of the DFT-based models shows that the processing time-dependence is more associated with the choice of the basis set than the exchange-correlation functional.

Mechanistic studies on Pyrobaculum calidifontis porphobilinogen synthase (5-aminolevulinic acid dehydratase).

Porphobilinogen synthase (PBG synthase) gene from Pyrobaculum calidifontis was cloned and expressed in E. coli. The recombinant enzyme was purified as an octamer and was found by mass spectrometry to have a subunit Mr of 37676.59 (calculated, 37676.3). The enzyme showed high thermal stability and retained almost all of its activity after incubation at 70 °C for 16 h in the presence of ß-mercaptoethanol (ß-ME) and zinc chloride. However, in the absence of the latter the enzyme was inactivated after 16 h although it regained full activity in the presence of ß-ME and zinc chloride. The protein contained 4 mol of tightly bound zinc per octamer. Further, 4 mol of low affinity zinc could be incorporated following incubation with exogenous zinc salts. The enzyme was inactivated by incubation with levulinic acid followed by treatment with sodium borohydride. Tryptic digest of the modified enzyme and mass spectrometric analysis showed that Lys257 was the site of modification, which has previously been shown to be the site for the binding of 5-aminolevulinic acid giving rise to the propionate-half of porphobilinogen. P. calidifontis PBG synthase was inactivated by 5-chlorolevulinic acid and the residue modified was shown to be the central cysteine (Cys127) of the zinc-binding cysteine-triad, comprising Cys125, 127, 135. The present results in conjunction with earlier findings on zinc containing PBG synthases, are discussed which advocate that the catalytic role of zinc in the activation of the 5-aminolevulinic acid molecule forming the acetate-half of PBG is possible.

Interaction of metals from group 10 (Ni, Pd, and Pt) and 11 (Cu, Ag, and Au) with human blood δ-ALA-D: in vitro and in silico studies.

Mammalian δ-aminolevulinate dehydratase (δ-ALA-D) is a metalloenzyme, which requires Zn(II) and reduced thiol groups for catalytic activity, and is an important molecular target for the widespread environmental toxic metals. The δ-ALA-D inhibition mechanism by metals of Group 10 (Ni, Pd, and Pt) and 11 (Cu, Ag, and Au) of the periodic table has not yet been determined. The objective of this study was to characterize the molecular mechanism of δ-ALA-D inhibition caused by the elements of groups 10 and 11 using in vitro (δ-ALA-D activity from human erythrocytes) and in silico (docking simulations) methods. Our results showed that Ni(II) and Pd(II) caused a small inhibition (~ 10%) of the δ-ALA-D. Pt(II) and Pt(IV) significantly inhibited the enzyme (75% and 44%, respectively), but this inhibition was attenuated by Zn(II) and dithiothreitol (DTT). In group 11, all metals inhibited δ-ALA-D with great potency (~ 70-90%). In the presence of Zn(II) and DTT, the enzyme activity was restored to the control levels. The in silico molecular docking data suggest that the coordination of the ions Pt(II), Pt(IV), Cu(II), Ag(I), and Au(III) with thiolates groups from C135 and C143 residues from the δ-ALA-D active site are crucial to the enzyme inhibition. The results indicate that a possible mechanism of inhibition of δ-ALA-D by these metals may involve the replacement of the Zn(II) from the active site and/or the cysteinyl residue oxidation.

The Remarkable Character of Porphobilinogen Synthase.

Porphobilinogen synthase (PBGS), also known as 5-aminolevulinate dehydratase, is an essential enzyme in the biosynthesis of all tetrapyrroles, which function in respiration, photosynthesis, and methanogenesis. Throughout evolution, PBGS adapted to a diversity of cellular niches and evolved to use an unusual variety of metal ions both for catalytic function and to control protein multimerization. With regard to the active site, some PBGSs require Zn2+; a subset of those, including human PBGS, contain a constellation of cysteine residues that acts as a sink for the environmental toxin Pb2+. PBGSs that do not require the soft metal ion Zn2+ at the active site instead are suspected of using the hard metal Mg2+. The most unexpected property of the PBGS family of enzymes is a dissociative allosteric mechanism that utilizes an equilibrium of architecturally and functionally distinct protein assemblies. The high-activity assembly is an octamer in which intersubunit interactions modulate active-site lid motion. This octamer can dissociate to dimer, the dimer can undergo a hinge twist, and the twisted dimer can assemble to a low-activity hexamer. The hexamer does not have the intersubunit interactions required to stabilize a closed conformation of the active site lid. PBGS active site chemistry benefits from a closed lid because porphobilinogen biosynthesis includes Schiff base formation, which requires deprotonated lysine amino groups. N-terminal and C-terminal sequence extensions dictate whether a specific species of PBGS can sample the hexameric assembly. The bulk of species (nearly all except animals and yeasts) use Mg2+ as an allosteric activator. Mg2+ functions allosterically by binding to an intersubunit interface that is present in the octamer but absent in the hexamer. This conformational selection allosteric mechanism is purported to be essential to avoid the untimely accumulation of phototoxic chlorophyll precursors in plants. For those PBGSs that do not use the allosteric Mg2+, there is a spatially equivalent arginine-derived guanidium group. Deprotonation of this residue promotes formation of the hexamer and accounts for the basic arm of the bell-shaped pH vs activity profile of human PBGS. A human inborn error of metabolism known as ALAD porphyria is attributed to PBGS variants that favor the hexameric assembly. The existence of one such variant, F12L, which dramatically stabilizes the human PBGS hexamer, allowed crystal structure determination for the hexamer. Without this crystal structure and octameric PBGS structures containing the allosteric Mg2+, it would have been difficult to decipher the structural basis for PBGS allostery. The requirement for multimer dissociation as an intermediate step in PBGS allostery was established by monitoring subunit disproportionation during the turnover-dependent transition of heteromeric PBGS (comprised of human wild type and F12L) from hexamer to octamer. One outcome of these studies was the definition of the dissociative morpheein model of protein allostery. The phylogenetically variable time scales for PBGS multimer interconversion result in atypical kinetic and biophysical behaviors. These behaviors can serve to identify other proteins that use the morpheein model of protein allostery.

Impact of quaternary structure dynamics on allosteric drug discovery.

The morpheein model of allosteric regulation draws attention to proteins that can exist as an equilibrium of functionally distinct assemblies where: one subunit conformation assembles into one multimer; a different subunit conformation assembles into a different multimer; and the various multimers are in a dynamic equilibrium whose position can be modulated by ligands that bind to a multimer-specific ligand binding site. The case study of porphobilinogen synthase (PBGS) illustrates how such an equilibrium holds lessons for disease mechanisms, drug discovery, understanding drug side effects, and identifying proteins wherein drug discovery efforts might focus on quaternary structure dynamics. The morpheein model of allostery has been proposed as applicable for a wide assortment of disease-associated proteins (Selwood, T., Jaffe, E., (2012) Arch. Bioch. Biophys, 519:131-143). Herein we discuss quaternary structure dynamics aspects to drug discovery for the disease-associated putative morpheeins phenylalanine hydroxylase, HIV integrase, pyruvate kinase, and tumor necrosis factor α. Also highlighted is the quaternary structure equilibrium of transthyretin and successful drug discovery efforts focused on controlling its quaternary structure dynamics.

Redox and metal-regulated oligomeric state for human porphobilinogen synthase activation.

The oligomeric state of human porphobilinogen synthase (PBGS) [EC.4.2.1.24] is homooctamer, which consists of conformationally heterogenous subunits in the tertiary structure under air-saturated conditions. When PBGS is activated by reducing agent with zinc ion, a reservoir zinc ion coordinated by Cys(223) is transferred in the active center to be coordinated by Cys(122), Cys(124), and Cys(132) (Sawada et al. in J Biol Inorg Chem 10:199-207, 2005). The latter zinc ion serves as an electrophilic catalysis. In this study, we investigated a conformational change associated with the PBGS activation by reducing agent and zinc ion using analytical ultracentrifugation, negative staining electron microscopy, native PAGE, and enzyme activity staining. The results are in good agreement with our notion that the main component of PBGS is octamer with a few percent of hexamer and that the octamer changes spatial subunit arrangement upon reduction and further addition of zinc ion, accompanying decrease in f/f (0). It is concluded that redox-regulated PBGS activation via cleavage of disulfide bonds among Cys(122), Cys(124), and Cys(132) and coordination with zinc ion is closely linked to change in the oligomeric state.

Isolation and biochemical characterization of delta-aminolevulinic acid dehydratase from Streptomyces yokosukanensis ATCC 25520.

In this study, delta-aminolevulinic acid dehydratase from Streptomyces yokosukanensis ATCC 25520, producer of an unusual purine riboside antibiotic called nebularine, was purified and characterized. Purification procedures involved with ammonium sulphate precipitation and gel filtration techniques by use of Sephacryl S-200. After gel filtration a 90.76-fold purification was obtained. The maximum enzymic activity was observed in the supernatant after 100% precipitation. According to the data obtained from investigation, the enzyme was found to be a single polypeptide having molecular mass around 34.8 kDa. This was determined by SDS-PAGE. Its optimal temperature around 45 degrees C, and optimal pH was found to be 8.0. Some heavy metals, Pb2+, Zn2+, Fe3+, Co2+, Mn2+, Mg2+ inhibited its activity between 20-51%, Ni2+ increased its activity up to 15%.

The activation mechanism of human porphobilinogen synthase by 2-mercaptoethanol: intrasubunit transfer of a reserve zinc ion and coordination with three cysteines in the active center.

Human porphobilinogen synthase [EC.4.2.1.24] is a homo-octamer enzyme. In the active center of each subunit, four cysteines are titrated with 5,5'-dithiobis(2-nitrobenzoic acid). Cys(122), Cys(124) and Cys(132) are placed near two catalytic sites, Lys(199) and Lys(252), and coordinate a zinc ion, referred to as "a proximal zinc ion", and Cys(223) is placed at the orifice of the catalytic cavity and coordinates a zinc ion, referred to as "a distal zinc ion", with His(131) . When the wild-type enzymes C122A (Cys(122)-->Ala), C124A (Cys(124)-->Ala), C132A (Cys(132)-->Ala) and C223A (Cys(223)-->Ala) were oxidized by hydrogen peroxide, the levels of activity were decreased. Two cysteines were titrated with 5,5'-dithiobis(2-nitrobenzoic acid) in the wild-type enzyme, while on the other hand, one cysteine was titrated in the mutant enzymes. When wild-type and mutant enzymes were reduced by 2-mercaptoethanol, the levels of activity were increased: four and three cysteines were titrated, respectively, suggesting that a disulfide bond was formed among Cys(122), Cys(124) and Cys(132) under oxidizing conditions. We analyzed the enzyme-bound zinc ion of these enzymes using inductively coupled plasma mass spectrometry with gel-filtration chromatography. The results for C223A showed that the number of proximal zinc ions correlated to the level of enzymatic activity. Furthermore, zinc-ion-free 2-mercaptoethanol increased the activity of the wild-type enzyme without a change in the total number of zinc ions, but C223A was not activated. These findings suggest that a distal zinc ion moved to the proximal binding site when a disulfide bond among Cys(122), Cys(124) and Cys(132) was reduced by reductants. Thus, in the catalytic functioning of the enzyme, the distal zinc ion does not directly contribute but serves rather as a reserve as the next proximal one that catalyzes the enzyme reaction. A redox change of the three cysteines in the active center accommodates the catch and release of the reserve distal zinc ion placed at the orifice of the catalytic cavity.

Tracking the evolution of porphobilinogen synthase metal dependence in vitro.

Metal ions are indispensable cofactors for chemical catalysis by a plethora of enzymes. Porphobilinogen synthases (PBGSs), which catalyse the second step of tetrapyrrole biosynthesis, are grouped according to their dependence on Zn(2+). Using site-directed mutagenesis, we embarked on transforming Zn(2+)-independent Pseudomonas aeruginosa PBGS into a Zn(2+)-dependent enzyme. Nine PBGS variants were generated by permutationally introducing three cysteine residues and a further two residues into the active site of the enzyme to match the homologous Zn(2+)-containing PBGS from Escherichia coli. Crystal structures of seven enzyme variants were solved to elucidate the nature of Zn(2+) coordination at high resolution. The three single-cysteine variants were invariably found to be enzymatically inactive and only one (D139C) was found to bind detectable amounts of Zn(2+). The double mutant A129C/D139C is enzymatically active and binds Zn(2+) in a tetrahedral coordination. Structurally and functionally it mimics mycobacterial PBGS, which bears an equivalent Zn(2+)-coordination site. The remaining two double mutants, without known natural equivalents, reveal strongly distorted tetrahedral Zn(2+)-binding sites. Variant A129C/D131C possesses weak PBGS activity while D131C/D139C is inactive. The triple mutant A129C/D131C/D139C, finally, displays an almost ideal tetrahedral Zn(2+)-binding geometry and a significant Zn(2+)-dependent enzymatic activity. Two additional amino acid exchanges further optimize the active site architecture towards the E.coli enzyme with an additional increase in activity. Our study delineates the potential evolutionary path between Zn(2+)-free and Zn(2+)-dependent PBGS enyzmes showing that the rigid backbone of PBGS enzymes is an ideal framework to create or eliminate metal dependence through a limited number of amino acid exchanges.

Photodynamic therapy of head and neck tumors and non-tumor like disorders.

In conclusion, we would like to emphasize that photodynamic therapy can be used as a convenient and promising tool in the treatment of various malignancies in the head and neck area. It has to be stressed that PDT is still less effective in the treatment of more advanced cases of head and neck cancer. As the method of treatment which improves the quality of life, its usefulness can be considered as well as palliative treatment of non-operative cases. Although the method is not free of several side effects, such skin phototoxicity, burning, slight pain and edema in the irradiated location. All these side effects are transient and usually they disappear within 24 hours. The photodynamic treatment always results in excellent cosmetic effects without scarring or marring which are usually encountered after routine surgical operation.

Delta-aminolevulinic acid dehydratase from Plasmodium falciparum: indigenous versus imported.

The heme biosynthetic pathway of the malaria parasite is a drug target and the import of host delta-aminolevulinate dehydratase (ALAD), the second enzyme of the pathway, from the red cell cytoplasm by the intra erythrocytic malaria parasite has been demonstrated earlier in this laboratory. In this study, ALAD encoded by the Plasmodium falciparum genome (PfALAD) has been cloned, the protein overexpressed in Escherichia coli, and then characterized. The mature recombinant enzyme (rPfALAD) is enzymatically active and behaves as an octamer with a subunit Mr of 46,000. The enzyme has an alkaline pH optimum of 8.0 to 9.0. rPfALAD does not require any metal ion for activity, although it is stimulated by 20-30% upon addition of Mg2+. The enzyme is inhibited by Zn2+ and succinylacetone. The presence of PfALAD in P. falciparum can be demonstrated by Western blot analysis and immunoelectron microscopy. The enzyme has been localized to the apicoplast of the malaria parasite. Homology modeling studies reveal that PfALAD is very similar to the enzyme species from Pseudomonas aeruginosa, but manifests features that are unique and different from plant ALADs as well as from those of the bacterium. It is concluded that PfALAD, while resembling plant ALADs in terms of its alkaline pH optimum and apicoplast localization, differs in its Mg2+ independence for catalytic activity or octamer stabilization. Expression levels of PfALAD in P. falciparum, based on Western blot analysis, immunoelectron microscopy, and EDTA-resistant enzyme activity assay reveals that it may account for about 10% of the total ALAD activity in the parasite, the rest being accounted for by the host enzyme imported by the parasite. It is proposed that the role of PfALAD may be confined to heme synthesis in the apicoplast that may not account for the total de novo heme biosynthesis in the parasite.

Investigations on the metal switch region of human porphobilinogen synthase.

Porphobilinogen synthase (PBGS) is an ancient and highly conserved protein that functions in the first common step in tetrapyrrole biosynthesis. The PBGS protein sequence contains a unique metal switch region that has been postulated to dictate an exclusive catalytic use of either zinc or magnesium, and perhaps also potassium. In some PBGS, the cysteines of the metal switch sequence DXCXCX(Y/F)X(3)G(H/Q)CG have been demonstrated to bind a catalytic zinc, and in other PBGS, the aspartic acid residues of the metal switch sequence DXALDX(Y/F)X(3)G(H/Q)DG have been postulated to bind a catalytically essential magnesium and/or potassium. The current work describes chimeric proteins that contain the aspartate-rich sequences of pea PBGS and Pseudomonas aeruginosa PBGS in place of the naturally occurring cysteine-rich sequence of human PBGS. The resultant chimeric PBGS proteins, peainhuman PBGS and psuinhuman PBGS, are substantially activated by both magnesium and potassium, but not by zinc. The specific activities of the chimeras are significantly lower than human PBGS. Detailed kinetic and inhibition data are presented for both chimeric proteins and are discussed in terms of this unique phylogenetic variation in metal ion usage. The identity of a basic residue, which is Arg221 in human PBGS, strictly correlates with the presence or absence of the cysteine-rich sequence. Those PBGS with the aspartate-rich metal switch sequence contain Lys in the analogous position. The R221K mutation was inserted into wild type and chimeric human PBGS and found to further reduce the activity of both, illustrating the subtle nature of the role of this residue.

5-Aminolaevulinic acid dehydratase: metals, mutants and mechanism.

5-Aminolaevulinic acid dehydratase catalyses the formation of porphobilinogen from two molecules of 5-aminolaevulinic acid. The studies described highlight the importance of a bivalent metal ion and two active-site lysine residues for the functioning of 5-aminolaevulinic acid dehydratase. Dehydratases fall into two main categories: zinc-dependent enzymes and magnesium-dependent enzymes. Mutations that introduced zinc-binding ligands into a magnesium-dependent enzyme conferred an absolute requirement for zinc. Mutagenesis of lysine residues 247 and 195 in the Escherichia coli enzyme lead to dramatic effects on enzyme activity, with lysine 247 being absolutely essential. Mutation of either lysine 247 or 195 to cysteine, and treatment of the mutant enzyme with 2-bromethylamine, resulted in the recovery of substantial enzyme activity. The effects of the site-directed alkylating inhibitor, 5-chlorolaevulinic acid, and 4,7-dioxosebacic acid, a putative intermediate analogue, were investigated by X-ray crystallography. These inhibitors reacted with both active-site lysine residues. The role of these two lysine residues in the enzyme mechanism is discussed.

The x-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with substrate and three inhibitors.

The structures of 5-aminolaevulinic acid dehydratase (ALAD) complexed with substrate (5-aminolaevulinic acid) and three inhibitors: laevulinic acid, succinylacetone and 4-keto-5-aminolaevulinic acid, have been solved at high resolution. The ligands all bind by forming a covalent link with Lys263 at the active site. The structures define the interactions made by one of the two substrate moieties that bind to the enzyme during catalysis. All of the inhibitors induce a significant ordering of the flap covering the active site. Succinylacetone appears to be unique by inducing a number of conformational changes in loops covering the active site, which may be important for understanding the co-operative properties of ALAD enzymes. Succinylacetone is produced in large amounts by patients suffering from the hereditary disease type I tyrosinaemia and its potent inhibition of ALAD also has implications for the pathology of this disease. The most intriguing result is that obtained with 4-keto-5-amino-hexanoic acid, which seems to form a stable carbinolamine intermediate with Lys263. It appears that we have defined the structure of an intermediate of Schiff base formation that the substrate forms upon binding to the P-site of the enzyme.

The molecular mechanism of lead inhibition of human porphobilinogen synthase.

Human porphobilinogen synthase (PBGS) is a main target in lead poisoning. Human PBGS purifies with eight Zn(II) per homo-octamer; four ZnA have predominantly nonsulfur ligands, and four ZnB have predominantly sulfur ligands. Only four Zn(II) are required for activity. To better elucidate the roles of Zn(II) and Pb(II), we produced human PBGS mutants that are designed to lack either the ZnA or ZnB sites. These proteins, MinusZnA (H131A, C223A) and MinusZnB (C122A, C124A, C132A), each become purified with four Zn(II) per octamer, thus confirming an asymmetry in the human PBGS structure. MinusZnA is fully active, whereas MinusZnB is far less active, verifying an important catalytic role for ZnB and the removed cysteine residues. Kinetic properties of the mutants and wild type proteins are described. Comparison of Pb(II) inhibition of the mutants shows that ligands to both ZnA and ZnB interact with Pb(II). The ZnB ligands preferentially interact with Pb(II). At least one ZnA ligand is responsible for the slow tight binding behavior of Pb(II). The data support a novel model where a high affinity lead site is a hybrid of the ZnA and ZnB sites. We propose that the lone electron pair of Pb(II) precludes Pb(II) to function in PBGS catalysis.

The Schiff base complex of yeast 5-aminolaevulinic acid dehydratase with laevulinic acid.

The X-ray structure of the complex formed between yeast 5-aminolaevulinic acid dehydratase (ALAD) and the inhibitor laevulinic acid has been determined at 2.15 A resolution. The inhibitor binds by forming a Schiff base link with one of the two invariant lysines at the catalytic center: Lys263. It is known that this lysine forms a Schiff base link with substrate bound at the enzyme's so-called P-site. The carboxyl group of laevulinic acid makes hydrogen bonds with the side-chain-OH groups of Tyr329 and Ser290, as well as with the main-chain >NH group of Ser290. The aliphatic moiety of the inhibitor makes hydrophobic interactions with surrounding aromatic residues in the protein including Phe219, which resides in the flap covering the active site. Our analysis strongly suggests that the same interactions will be made by P-side substrate and also indicates that the substrate that binds at the enzyme's A-site will interact with the enzyme's zinc ion bound by three cysteines (133, 135, and 143). Inhibitor binding caused a substantial ordering of the active site flap (residues 217-235), which was largely invisible in the native electron density map and indicates that this highly conserved yet flexible region has a specific role in substrate binding during catalysis.

X-ray structure of 5-aminolevulinic acid dehydratase from Escherichia coli complexed with the inhibitor levulinic acid at 2.0 A resolution.

5-Aminolevulinic acid dehydratase (ALAD), an early enzyme of the tetrapyrrole biosynthesis pathway, catalyzes the dimerization of 5-aminolevulinic acid to form the pyrrole, porphobilinogen. ALAD from Escherichia coli is shown to form a homo-octameric structure with 422 symmetry in which each subunit adopts the TIM barrel fold with a 30-residue N-terminal arm. Pairs of monomers associate with their arms wrapped around each other. Four of these dimers interact, principally via their arm regions, to form octamers in which each active site is located on the surface. The active site contains two lysine residues (195 and 247), one of which (Lys 247) forms a Schiff base link with the bound substrate analogue, levulinic acid. Of the two substrate binding sites (referred to as A and P), our analysis defines the residues forming the P-site, which is where the first ALA molecule to associate with the enzyme binds. The carboxyl group of the levulinic acid moiety forms hydrogen bonds with the side chains of Ser 273 and Tyr 312. In proximity to the levulinic acid is a zinc binding site formed by three cysteines (Cys 120, 122, and 130) and a solvent molecule. We infer that the second substrate binding site (or A-site) is located between the triple-cysteine zinc site and the bound levulinic acid moiety. Two invariant arginine residues in a loop covering the active site (Arg 205 and Arg 216) appear to be appropriately placed to bind the carboxylate of the A-site substrate. Another metal binding site, close to the active site flap, in which a putative zinc ion is coordinated by a carboxyl and five solvent molecules may account for the activating properties of magnesium ions.

delta-Aminolevulinate dehydratase inhibition by ascorbic acid is mediated by an oxidation system existing in the hepatic supernatant.

The effect of ascorbic acid (AA) on hepatic delta-aminolevulinic acid dehydratase (ALA-D) activity was studied. AA decreased enzyme activity by reducing maximum velocity and tended to increase the Michaelis constant. ALA-D inactivation by AA occurred similarly both in air and argonium atmosphere incubation. DTT reduced considerably the inhibitory effect of AA on ALA-D, but glutathione was ineffective in reversing inactivation. These data indicate that inhibition occurs mainly due to an acceleration of the oxidation rate mediated by the hepatic supernatant utilizing AA in sulfhydryl groups of cysteine residues present at the ALA-D active site. AA probably acts on cysteine from the ALA-D B site since cucumber and radish leaves ALA-D was not inhibited by AA (up to 16 mM). The addition of free radical scavengers to the medium did not alter ALA-D inactivation caused by AA, indicating that active oxygen species formed during AA oxidation were not directly related to -SH oxidation. The chelation of zinc ions from the enzyme by EDTA turned ALA-D more susceptible to the inhibitory effect of AA. This effect seems to involve mainly ZnB, which is known to bind to four cysteines. The present data suggest that AA may participate in the regulation of the heme biosynthesis pathway by promoting a reversible inactivation of ALA-D.