X-ray Structure of 5-Aminolevulinic Acid Dehydratase from <i>Escherichia coli</i> Complexed with the Inhibitor Levulinic Acid at 2.0 Å Resolution

Erskine, P.T.; Norton, E.; Cooper, J.B.; Lambert, Richard; Coker, Alun R.; Lewis, Gareth R.; Spencer, P.S.; Sarwar, Mohammed G.; Wood, Steve; Warren, Martin J.; Shoolingin‐Jordan, Peter M.

doi:10.1021/bi982137w

Cited by 92 publications

(101 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thus, Schiff base formation with ALA is not responsible for a conformational change that dictates half-site reactivity. This is consistent with crystallographic data that show Schiff base formation to levulinic acid does not cause protein asymmetry (7,9). It remains possible that binding of the C-5 amino group of P-side ALA, which is the ALA that forms the Schiff base, is essential for the evolution of the asymmetric structure.…”

Section: Mass Spectral Analysis Of Peptides From Wild-type E Coli Pbsupporting

confidence: 89%

“…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).…”

Section: Mass Spectral Analysis Of Peptides From Wild-type E Coli Pbsupporting

confidence: 85%

“…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.…”

Section: Nmr Studies Of Mutant and Wild-type Pbgs Using [4-mentioning

confidence: 99%

“…Yet much of the chemical reaction mechanism remains speculative (7). Only one intermediate, an enzyme-substrate Schiff base, has been identified (11).…”

mentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

See 4 more Smart Citations

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

Mitchell¹,

Volin

Martins

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

Porphobilinogen synthase (PBGS) is a homo-octameric protein that catalyzes the complex asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA). The only characterized intermediate in the PBGS-catalyzed reaction is a Schiff base that forms between the first ALA that binds and a conserved lysine, which in Escherichia coli PBGS is Lys-246 and in human PBGS is Lys-252. In this study, E. coli PBGS mutants K246H, K246M, K246W, K246N, and K246G and human PBGS mutant K252G were characterized. Alterations to this lysine result in a disabled but not totally inactive protein suggesting an alternate mechanism in which proximity and orientation are major catalytic devices. 13C NMR studies of [3,5-13 C]porphobilinogen bound at the active sites of the E. coli PBGS and the mutants show only minor chemical shift differences, i.e. environmental alterations. Mammalian PBGS is established to have four functional active sites, whereas the crystal structure of E. coli PBGS shows eight spatially distinct and structurally equivalent subunits. Biochemical data for E. coli PBGS have been interpreted to support both four and eight active sites. A unifying hypothesis is that formation of the Schiff base between this lysine and ALA triggers a conformational change that results in asymmetry. Product binding studies with wild-type E. coli PBGS and K246G demonstrate that both bind porphobilinogen at four per octamer although the latter cannot form the Schiff base from substrate. Thus, formation of the lysine to ALA Schiff base is not required to initiate the asymmetry that results in half-site reactivity.Porphobilinogen synthase (PBGS, 1 also known as 5-aminolevulinate dehydratase) is a metalloenzyme that catalyzes the asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA) to form porphobilinogen, the monopyrrole precursor of tetrapyrroles (see Fig. 1). Although PBGS proteins from different organisms differ in their use of metal ions, there is remarkable sequence conservation between the PBGS from eubacteria, archaea, and eucaryotes implying a commonality in the overall protein architecture and reaction mechanism (1). The two PBGS studied herein, those of Escherichia coli and human, both require a catalytic Zn(II), neither require monovalent cations, and the E. coli PBGS activity is stimulated by an allosteric Mg(II) (2-5). The overall protein sequence identity between E. coli and human PBGS is 42%, and the active site residues are significantly more conserved.Considerable effort has been applied to the characterization of PBGS proteins from various organisms, and several crystal structures are now available (6 -10). Yet much of the chemical reaction mechanism remains speculative (7). Only one intermediate, an enzyme-substrate Schiff base, has been identified (11). Several x-ray crystal structures contain levulinic acid bound in a fashion analogous to the Schiff base (Protein Database codes 1B4K, 1YLV, and 1B4E), and the actual Schiff base involving ALA has been observed by 13 C and 15 N NMR for a chemically mo...

show abstract

Section: Mass Spectral Analysis Of Peptides From Wild-type E Coli Pbsupporting

confidence: 89%

Section: Mass Spectral Analysis Of Peptides From Wild-type E Coli Pbsupporting

confidence: 85%

Section: Nmr Studies Of Mutant and Wild-type Pbgs Using [4-mentioning

confidence: 99%

“…Yet much of the chemical reaction mechanism remains speculative (7). Only one intermediate, an enzyme-substrate Schiff base, has been identified (11).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

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

Mitchell¹,

Volin

Martins

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

5‐Aminolaevulinic Acid Dehydratase

Cooper

Erskine

2004

Handbook of Metalloproteins

View full text Add to dashboard Cite

The enzyme 5‐aminolaevulinic acid dehydratase (ALAD, E.C.4.2.1.24), sometimes referred to as porphobilinogen synthase, catalyzes the second step in the biosynthesis of tetrapyrroles involving the condensation between two 5‐aminolaevulinic acid (ALA) molecules to form the pyrrole porphobilinogen (PBG). The X‐ray structures of several ALADs have been determined showing that the enzyme forms a large homo‐octameric structure with all eight active sites on the outer surface. Each subunit adopts the triose phosphate isomerase (TIM) barrel fold with an N‐terminal arm, which forms extensive intersubunit interactions. Pairs of monomers associate with their arms wrapped around each other to form compact dimers. Four dimers, which interact principally via their arm regions, form the octamer, which has a large solvent‐filled cavity in the center. The active site of each subunit is located in a pronounced cavity formed by loops at the C‐terminal ends of the strands forming the TIM barrel. In humans, hereditary deficiencies in ALAD give rise to the rare disease Doss porphyria, and the sensitivity of the enzyme to inhibition by lead ions is thought to be one of the main problems in acute lead poisoning. ALADs from many species (except plants and some bacteria) have a zinc ion at the active site, which has been implicated in the catalytic mechanism and is readily displaced by the inhibitor lead. These ALADs have a cysteine‐rich motif that coordinates the active site zinc ion (α‐site). In contrast, ALADs from plants and some bacteria have a number of mutations in the metal‐binding motif that replace the cysteine residues with other amino acids. Two of the cysteines are replaced by aspartate, which is thought to be more appropriate for coordination of magnesium ions, which these enzymes apparently require instead of zinc. Some ALADs have an additional zinc or magnesium binding site located between the subunits of the octamer (β‐site) that has more of a structural or activating role. Early inhibitor binding studies defined the interactions made by only one of the two substrate moieties (P‐side substrate) that bind to the enzyme during catalysis. Biochemical and crystallographic data showed that P‐side substrate binds by forming a Schiff base with the enzyme at Lys247 ( Escherichia coli ALAD numbering). Until very recently there has been little evidence that binding of the second substrate moiety (A‐side substrate) involves formation of a Schiff base with the enzyme. However, the structures of diacid inhibitors provided an improved definition of the interactions made by both the substrate molecules (A‐ and P‐side). The most intriguing result was the finding that 4,7‐dioxosebacic acid forms a second Schiff base with the enzyme involving Lys195 ( E. coli ALAD numbering), suggesting that A‐side substrate makes the same interaction during catalysis. This finding has been substantiated by the demonstration that the inhibitor 5‐fluorolaevulinic acid binds by making Schiff bases with both the active site lysine residues. Current proposals for the catalytic mechanism involve the binding of both substrate moieties by formation of Schiff bases with the active site lysines.

show abstract