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Conformational stability and activity analysis of two hydroxymethylbilane synthase mutants, K132N and V215E, with different phenotypic association with acute intermittent porphyria
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| Zeitschriftentitel: | Bioscience Reports |
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| Personen und Körperschaften: | , , , , , |
| In: | Bioscience Reports, 33, 2013, 4 |
| Format: | E-Article |
| Sprache: | Englisch |
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Portland Press Ltd.
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| author_facet |
Bustad, Helene J. Vorland, Marta Rønneseth, Eva Sandberg, Sverre Martinez, Aurora Toska, Karen Bustad, Helene J. Vorland, Marta Rønneseth, Eva Sandberg, Sverre Martinez, Aurora Toska, Karen |
|---|---|
| author |
Bustad, Helene J. Vorland, Marta Rønneseth, Eva Sandberg, Sverre Martinez, Aurora Toska, Karen |
| spellingShingle |
Bustad, Helene J. Vorland, Marta Rønneseth, Eva Sandberg, Sverre Martinez, Aurora Toska, Karen Bioscience Reports Conformational stability and activity analysis of two hydroxymethylbilane synthase mutants, K132N and V215E, with different phenotypic association with acute intermittent porphyria Cell Biology Molecular Biology Biochemistry Biophysics |
| author_sort |
bustad, helene j. |
| spelling |
Bustad, Helene J. Vorland, Marta Rønneseth, Eva Sandberg, Sverre Martinez, Aurora Toska, Karen 0144-8463 1573-4935 Portland Press Ltd. Cell Biology Molecular Biology Biochemistry Biophysics http://dx.doi.org/10.1042/bsr20130045 <jats:p>The autosomal dominantly inherited disease AIP (acute intermittent porphyria) is caused by mutations in HMBS [hydroxymethylbilane synthase; also known as PBG (porphobilinogen) deaminase], the third enzyme in the haem biosynthesis pathway. Enzyme-intermediates with increasing number of PBG molecules are formed during the catalysis of HMBS. In this work, we studied the two uncharacterized mutants K132N and V215E comparative with wt (wild-type) HMBS and to the previously reported AIP-associated mutants R116W, R167W and R173W. These mainly present defects in conformational stability (R116W), enzyme kinetics (R167W) or both (R173W). A combination of native PAGE, CD, DSF (differential scanning fluorimetry) and ion-exchange chromatography was used to study conformational stability and activity of the recombinant enzymes. We also investigated the distribution of intermediates corresponding to specific elongation stages. It is well known that the thermostability of HMBS increases when the DPM (dipyrromethane) cofactor binds to the apoenzyme and the holoenzyme is formed. Interestingly, a decrease in thermal stability was measured concomitant to elongation of the pyrrole chain, indicating a loosening of the structure prior to product release. No conformational or kinetic defect was observed for the K132N mutant, whereas V215E presented lower conformational stability and probably a perturbed elongation process. This is in accordance with the high association of V215E with AIP. Our results contribute to interpret the molecular mechanisms for dysfunction of HMBS mutants and to establish genotype–phenotype relations for AIP.</jats:p> Conformational stability and activity analysis of two hydroxymethylbilane synthase mutants, K132N and V215E, with different phenotypic association with acute intermittent porphyria Bioscience Reports |
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| title |
Conformational stability and activity analysis of two hydroxymethylbilane synthase mutants, K132N and V215E, with different phenotypic association with acute intermittent porphyria |
| title_unstemmed |
Conformational stability and activity analysis of two hydroxymethylbilane synthase mutants, K132N and V215E, with different phenotypic association with acute intermittent porphyria |
| title_full |
Conformational stability and activity analysis of two hydroxymethylbilane synthase mutants, K132N and V215E, with different phenotypic association with acute intermittent porphyria |
| title_fullStr |
Conformational stability and activity analysis of two hydroxymethylbilane synthase mutants, K132N and V215E, with different phenotypic association with acute intermittent porphyria |
| title_full_unstemmed |
Conformational stability and activity analysis of two hydroxymethylbilane synthase mutants, K132N and V215E, with different phenotypic association with acute intermittent porphyria |
| title_short |
Conformational stability and activity analysis of two hydroxymethylbilane synthase mutants, K132N and V215E, with different phenotypic association with acute intermittent porphyria |
| title_sort |
conformational stability and activity analysis of two hydroxymethylbilane synthase mutants, k132n and v215e, with different phenotypic association with acute intermittent porphyria |
| topic |
Cell Biology Molecular Biology Biochemistry Biophysics |
| url |
http://dx.doi.org/10.1042/bsr20130045 |
| publishDate |
2013 |
| physical |
|
| description |
<jats:p>The autosomal dominantly inherited disease AIP (acute intermittent porphyria) is caused by mutations in HMBS [hydroxymethylbilane synthase; also known as PBG (porphobilinogen) deaminase], the third enzyme in the haem biosynthesis pathway. Enzyme-intermediates with increasing number of PBG molecules are formed during the catalysis of HMBS. In this work, we studied the two uncharacterized mutants K132N and V215E comparative with wt (wild-type) HMBS and to the previously reported AIP-associated mutants R116W, R167W and R173W. These mainly present defects in conformational stability (R116W), enzyme kinetics (R167W) or both (R173W). A combination of native PAGE, CD, DSF (differential scanning fluorimetry) and ion-exchange chromatography was used to study conformational stability and activity of the recombinant enzymes. We also investigated the distribution of intermediates corresponding to specific elongation stages. It is well known that the thermostability of HMBS increases when the DPM (dipyrromethane) cofactor binds to the apoenzyme and the holoenzyme is formed. Interestingly, a decrease in thermal stability was measured concomitant to elongation of the pyrrole chain, indicating a loosening of the structure prior to product release. No conformational or kinetic defect was observed for the K132N mutant, whereas V215E presented lower conformational stability and probably a perturbed elongation process. This is in accordance with the high association of V215E with AIP. Our results contribute to interpret the molecular mechanisms for dysfunction of HMBS mutants and to establish genotype–phenotype relations for AIP.</jats:p> |
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| author | Bustad, Helene J., Vorland, Marta, Rønneseth, Eva, Sandberg, Sverre, Martinez, Aurora, Toska, Karen |
| author_facet | Bustad, Helene J., Vorland, Marta, Rønneseth, Eva, Sandberg, Sverre, Martinez, Aurora, Toska, Karen, Bustad, Helene J., Vorland, Marta, Rønneseth, Eva, Sandberg, Sverre, Martinez, Aurora, Toska, Karen |
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| description | <jats:p>The autosomal dominantly inherited disease AIP (acute intermittent porphyria) is caused by mutations in HMBS [hydroxymethylbilane synthase; also known as PBG (porphobilinogen) deaminase], the third enzyme in the haem biosynthesis pathway. Enzyme-intermediates with increasing number of PBG molecules are formed during the catalysis of HMBS. In this work, we studied the two uncharacterized mutants K132N and V215E comparative with wt (wild-type) HMBS and to the previously reported AIP-associated mutants R116W, R167W and R173W. These mainly present defects in conformational stability (R116W), enzyme kinetics (R167W) or both (R173W). A combination of native PAGE, CD, DSF (differential scanning fluorimetry) and ion-exchange chromatography was used to study conformational stability and activity of the recombinant enzymes. We also investigated the distribution of intermediates corresponding to specific elongation stages. It is well known that the thermostability of HMBS increases when the DPM (dipyrromethane) cofactor binds to the apoenzyme and the holoenzyme is formed. Interestingly, a decrease in thermal stability was measured concomitant to elongation of the pyrrole chain, indicating a loosening of the structure prior to product release. No conformational or kinetic defect was observed for the K132N mutant, whereas V215E presented lower conformational stability and probably a perturbed elongation process. This is in accordance with the high association of V215E with AIP. Our results contribute to interpret the molecular mechanisms for dysfunction of HMBS mutants and to establish genotype–phenotype relations for AIP.</jats:p> |
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| spelling | Bustad, Helene J. Vorland, Marta Rønneseth, Eva Sandberg, Sverre Martinez, Aurora Toska, Karen 0144-8463 1573-4935 Portland Press Ltd. Cell Biology Molecular Biology Biochemistry Biophysics http://dx.doi.org/10.1042/bsr20130045 <jats:p>The autosomal dominantly inherited disease AIP (acute intermittent porphyria) is caused by mutations in HMBS [hydroxymethylbilane synthase; also known as PBG (porphobilinogen) deaminase], the third enzyme in the haem biosynthesis pathway. Enzyme-intermediates with increasing number of PBG molecules are formed during the catalysis of HMBS. In this work, we studied the two uncharacterized mutants K132N and V215E comparative with wt (wild-type) HMBS and to the previously reported AIP-associated mutants R116W, R167W and R173W. These mainly present defects in conformational stability (R116W), enzyme kinetics (R167W) or both (R173W). A combination of native PAGE, CD, DSF (differential scanning fluorimetry) and ion-exchange chromatography was used to study conformational stability and activity of the recombinant enzymes. We also investigated the distribution of intermediates corresponding to specific elongation stages. It is well known that the thermostability of HMBS increases when the DPM (dipyrromethane) cofactor binds to the apoenzyme and the holoenzyme is formed. Interestingly, a decrease in thermal stability was measured concomitant to elongation of the pyrrole chain, indicating a loosening of the structure prior to product release. No conformational or kinetic defect was observed for the K132N mutant, whereas V215E presented lower conformational stability and probably a perturbed elongation process. This is in accordance with the high association of V215E with AIP. Our results contribute to interpret the molecular mechanisms for dysfunction of HMBS mutants and to establish genotype–phenotype relations for AIP.</jats:p> Conformational stability and activity analysis of two hydroxymethylbilane synthase mutants, K132N and V215E, with different phenotypic association with acute intermittent porphyria Bioscience Reports |
| spellingShingle | Bustad, Helene J., Vorland, Marta, Rønneseth, Eva, Sandberg, Sverre, Martinez, Aurora, Toska, Karen, Bioscience Reports, Conformational stability and activity analysis of two hydroxymethylbilane synthase mutants, K132N and V215E, with different phenotypic association with acute intermittent porphyria, Cell Biology, Molecular Biology, Biochemistry, Biophysics |
| title | Conformational stability and activity analysis of two hydroxymethylbilane synthase mutants, K132N and V215E, with different phenotypic association with acute intermittent porphyria |
| title_full | Conformational stability and activity analysis of two hydroxymethylbilane synthase mutants, K132N and V215E, with different phenotypic association with acute intermittent porphyria |
| title_fullStr | Conformational stability and activity analysis of two hydroxymethylbilane synthase mutants, K132N and V215E, with different phenotypic association with acute intermittent porphyria |
| title_full_unstemmed | Conformational stability and activity analysis of two hydroxymethylbilane synthase mutants, K132N and V215E, with different phenotypic association with acute intermittent porphyria |
| title_short | Conformational stability and activity analysis of two hydroxymethylbilane synthase mutants, K132N and V215E, with different phenotypic association with acute intermittent porphyria |
| title_sort | conformational stability and activity analysis of two hydroxymethylbilane synthase mutants, k132n and v215e, with different phenotypic association with acute intermittent porphyria |
| title_unstemmed | Conformational stability and activity analysis of two hydroxymethylbilane synthase mutants, K132N and V215E, with different phenotypic association with acute intermittent porphyria |
| topic | Cell Biology, Molecular Biology, Biochemistry, Biophysics |
| url | http://dx.doi.org/10.1042/bsr20130045 |