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Acute intermittent porphyria in Sweden. Molecular, functional and clinical consequences of some new mutations found in the porphobilinogen deaminase gene
No full textAcute intermittent porphyria (AIP) is an autosomal dominant disorder caused by a partial deficit of porphobilinogen deaminase (PBGD), the third of eight enzymes in the haem biosynthetic pathway. The overt disease is characterized by neuropsychiatric symptoms that are often triggered by exogenous factors such as certain drugs, stress, and alcohol. The aim of this work has been to identify the underlying genetic defect in each AIP-affected family in order to provide early counselling to assist in the avoidance of precipitating factors. The prevalence of AIP in Sweden is in the order of 1:10 000. The major mutation in Sweden, W198X, is due to a founder effect in the northern part of the country. This mutation, together with a further 11 mutations, have been reported previously. The present communication encompasses the great majority of AIP kindreds in Sweden and includes a further 27 mutations within the PBGD gene. This includes 14 completely new mutations, as well as 11 known mutations detected for the first time in Sweden. The majority of the mutations are located in exons 10 and 12 with fewer in exon 7. The clinical and biochemical outcomes in some patients are described. We also use the three-dimensional structure of the porphobilinogen deaminase enzyme to predict the possible molecular and functional consequences of the new Swedish missense and nonsense mutations
5-Aminolaevulinic acid dehydratase: metals, mutants and mechanism
No full text5-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
Structure of Chlorobium vibrioforme 5-aminolaevulinic acid dehydratase complexed with a diacid inhibitor
No full textThe structure of Chlorobium vibrioforme 5-aminolaevulinic acid dehydratase (ALAD) complexed with the irreversible inhibitor 4,7-dioxosebacic acid has been solved. The inhibitor binds by forming Schiff-base linkages with lysines 200 and 253 at the active site. The structure reported here provides a definition of the interactions made by both of the substrate molecules (A-side and P-side substrates) with the C. vibrioforme ALAD and is compared and contrasted with structures of the same inhibitor bound to Escherichia coli and yeast ALAD. The structure suggests why 4,7-dioxosebacic acid is a better inhibitor of the zinc-dependent ALADs than of the zinc-independent ALADs
Human porphobilinogen deaminase mutations in the investigation of the mechanism of dipyrromethane cofactor assembly and tetrapyrrole formation
No full textPorphobilinogen deaminase mutants that cause acute intermittent porphyria have been investigated as recombinant proteins expressed in Escherichia coli, yielding important insight into the mechanism of dipyrromethane cofactor assembly and tetrapyrrole chain polymerization. A mutation that affects a key catalytic residue, D99G, results in an inactive holo-protein that exists as a complex with two substrate molecules covalently bound to the dipyrromethane cofactor arising from the reaction between the apo-protein and pre-uroporphyrinogen. The R149Q mutant is also devoid of catalytic activity but the mutant protein is unable to assemble the dipyrromethane cofactor from pre-uroporphyrinogen and persists as an unstable, heat-labile apo-protein. The mutant, R173Q, has very low activity and, like R149Q, also exhibits largely as an apo-protein. The inability to reconstitute either R149Q or R173Q with exogenous pre-uroporphyrinogen confirms the importance of these two arginine residues for dipyrromethane cofactor assembly. In contrast, the mutant R167Q exists as a holo-enzyme but the catalytic cycle is severely compromised, leading to the accumulation of stable enzyme–substrate intermediates from the catalytic cycle
X-ray structure of a putative reaction intermediate of 5-aminolaevulinic acid dehydratase
No full textThe X-ray structure of yeast 5-aminolaevulinic acid dehydratase, in which the catalytic site of the enzyme is complexed with a putative cyclic intermediate composed of both substrate moieties, has been solved at 0.16 nm (1.6 Å) resolution. The cyclic intermediate is bound covalently to Lys(263) with the amino group of the aminomethyl side chain ligated to the active-site zinc ion in a position normally occupied by a catalytic hydroxide ion. The cyclic intermediate is catalytically competent, as shown by its turnover in the presence of added substrate to form porphobilinogen. The findings, combined with those of previous studies, are consistent with a catalytic mechanism in which the C–C bond linking both substrates in the intermediate is formed before the C–N bond
The x-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with substrate and three inhibitors
No full textThe 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 byone 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 numberof 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-aminohexanoic acid, which seems to form a stable carbinolamine intermediate with Lys263. It appears that we have defined the structure of an intermediateof Schiff base formation that the substrate forms upon binding to the P-site of the enzyme
5-Aminolevulinic acid synthase: mechanism, mutations and medicine
No full text5-Aminolevulinic acid synthase (ALAS), the first enzyme of the heme biosynthesis pathway, catalyses the pyridoxal 5'-phosphate-dependent condensation between glycine and succinyl-CoA to yield 5-aminolevulinic acid (5-amino-4-oxopentanoate). A three-dimensional structural model of Rhodobacter spheroides ALAS has been constructed and used to identify amino acid residues at the active site that are likely to be important for the recognition of glycine, the only amino acid substrate. Several residues have been investigated by site-directed mutagenesis and enzyme variants have been generated that are able to use alanine, serine or threonine. A three-dimensional structure model of 5-aminolevulinic acid synthase from human erythrocytes (ALAS 2) has also been constructed and used to map a range of naturally occurring human mutants that give rise to X-linked sideroblastic anemia. A number of these anemias respond favourably to vitamin B(6) (pyridoxine) therapy, whereas others are either partially responsive or completely refractory. Detailed investigations with selected human mutants have highlighted the importance of arginine-517 that is implicated in glycine carboxyl group binding
X-Ray structure of porphobilinogen deaminase from A. Thaliana at 1.5A resolution
No full textThe enzyme, porphobilinogen deaminase (PBGD), catalyses the stepwise polymerization of themonopyrrole, porphobilinogen (PBG), to give the linear hydroxymethylbilane,preuroporphyrinogen. Preuroporphyrinogen is then cyclised, with rearrangement, to giveuroporphyrinogen III, the ubiquitous precursor of haems, chlorophylls and corrins. InArabidopsis thaliana, PBGD is the fifth enzyme of the chlorophyll biosynthetic pathway.The mature A. thaliana PBGD protein of 320 amino acids was expressed from two syntheticgenes using the pT7-7 vector in Escherichia coli strain BL21 (DE3). One construct wasidentical to the nucleotide sequence of the A. thaliana HEMC (AT5G08280) coding region andthe other was similar, but with an E. coli codon bias. Neither recombinant enzyme contained thechloroplast import sequence and possessed N-termini of NH2-MVAV… rather than NH2-CVAV… A high proportion (over 90%) of the expressed protein was found to be insoluble andmuch time was spent increasing the yield of soluble enzyme and obtaining sufficient material forcrystal preparation. No significant differences in expression were noted for the two constructsand both purified enzymes had Mr values of 34,928 ± 4 as measured by mass spectrometry.Crystals were obtained from screens using 30% PEG 4000, 0.1M NaAc and 0.1M MgCl2, pH4.6, containing 2mM dithothreitol. Data from suitable crystals were collected at the ESRF atGrenoble and were in space group C2 with unit cell dimensions of 142.1Å x 37.36Å x 55.37Å;?=90.00º, ?=104.83º and ?=90.00º. A structure for the A. thaliana PBGD was obtained at 1.5Åresolution by molecular replacement using the E. coli PBDG enzyme as search model.Programmes used for the refinement included the CCP4 suite, MOSFLM, SORTMTZ, SCALA,TRUNCATE and HKL VIEW.The structure of the A. thaliana PBGD enzyme shows the presence of three domains, each ofapproximately 100 residues. A deep cavity between domains I and II constitutes the active siteand harbours the dipyrromethane cofactor. Domain III provides the attachment site for thecofactor which is covalently bound to Cys 254. The structure shows, for the first time, a flap or“lid” over the active site, not previously observed in the E. coli and human PBGD structures.The differences and similarities between the A. thaliana PBGD structure and deaminasestructures from E. coli and human sources are discussed. As security, a selenomethioninederivative of the enzyme was also prepared and crystals were obtained for possiblemultiwavelength anomalous dispersion (MAD) experiments.Two mutants of A. thaliana PBGD, D95N and R161K, were prepared and the proteins wereisolated. The D95N mutation led to an inactive enzyme, whereas the R161K mutation yieldedan enzyme with 10% activity, and a lowered pH optimum, since the mutation substituted one ofthe six conserved active site arginine residues.The thesis presents, for the first time, the X-ray structure of a PBGD from a higher plant,Arabidopsis thaliana, and is the first time that the “lid” over the active site has been resolved.The importance of the active site “lid” in the functioning of the enzyme is discussed
The X-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with two diacid inhibitors
No full textThe structures of 5-aminolaevulinic acid dehydratase complexed with two irreversible inhibitors (4-oxosebacic acid and 4,7-dioxosebacic acid) have been solved at high resolution. Both inhibitors bind by forming a Schiff base link with Lys 263 at the active site. Previous inhibitor binding studies have defined the interactions made by only one of the two substrate moieties (P-side substrate) which bind to the enzyme during catalysis. The structures reported here provide an improved definition of the interactions made by both of the substrate molecules (A- and P-side substrates). The most intriguing result is the novel finding that 4,7-dioxosebacic acid forms a second Schiff base with the enzyme involving Lys 210. It has been known for many years that P-side substrate forms a Schiff base (with Lys 263) but until now there has been no evidence that binding of A-side substrate involves formation of a Schiff base with the enzyme. A catalytic mechanism involving substrate linked to the enzyme through Schiff bases at both the A- and P-sites is proposed.<br/
Structure of yeast 5-aminolaevulinic acid dehydratase complexed with the inhibitor 5-hydroxylaevulinic acid
Get PDFThe X-ray structure of the enzyme 5-aminolaevulinic acid dehydratase (ALAD) from yeast complexed with the competitive inhibitor 5-hydroxylaevulinic acid has been determined at a resolution of 1.9 Å . The structure shows that the inhibitor is bound by a Schiff-base link to one of the invariant active-site lysine residues (Lys263). The inhibitor appears to bind in two well defined conformations and the interactions made by it suggest that it is a very close analogue of the substrate 5-aminolaevulinic acid (ALA)
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