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Crystal structure of human persulfide dioxygenase: structural basis of ethylmalonic encephalopathy.

Pettinati I, Brem J, McDonough MA, Schofield CJ - Hum. Mol. Genet. (2015)

Bottom Line: A channel leading to the active site is sufficiently large to accommodate a GSSH substrate.Some of the observed hETHE1 clinical mutations cluster in the active site region.The structure will serve as a basis for detailed functional and mechanistic studies on ETHE1 and will be useful in the development of selective MBL inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK.

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hETHE1 activity assay and oligomerization state. (A) Oxygen consumption activity assay. hETHE1 activity was measured as percentage of oxygen consumed in the presence of GSSH. Each sample was performed in triplicate. (B) Non-denaturing electrospray soft-ionization mass spectrometry deconvoluted spectrum of purified hETHE1 protein indicates that hETHE1 is primarily dimeric. Peak A (26 170 Da) represents monomer; peak B (52370 Da) the dimer. In both monomeric and dimeric states one iron ion (+56 Da) is bound to each protomer while only in the dimer a +30 Da was observed possibly due to the oxidation state of Cys247 (see the main text and Supplementary Material, Fig. S5) (conditions: 15 µm of hETHE1 in 15 mm ammonium acetate buffer (pH 7.5); cone voltage for the acquisition of the spectra was 80 V). (C) MALS analysis of hETHE1 protein after purification. Peak A (∼51 560 Da) represents monomer; peak B (∼99 190 Da) the tetramer. MALS experiments were carried out by the Biophysical Services of the Biochemistry Department of Oxford University. (D) The molecular mass of hETHE1 in solution was estimated using a Sephadex G250 gel filtration column calibrated with protein standards [beta-amylase (223 kDa), albumin (66 kDa), carbonic anhydrase (29 kDa) and cytochrome C (12 kDa)].
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DDV007F3: hETHE1 activity assay and oligomerization state. (A) Oxygen consumption activity assay. hETHE1 activity was measured as percentage of oxygen consumed in the presence of GSSH. Each sample was performed in triplicate. (B) Non-denaturing electrospray soft-ionization mass spectrometry deconvoluted spectrum of purified hETHE1 protein indicates that hETHE1 is primarily dimeric. Peak A (26 170 Da) represents monomer; peak B (52370 Da) the dimer. In both monomeric and dimeric states one iron ion (+56 Da) is bound to each protomer while only in the dimer a +30 Da was observed possibly due to the oxidation state of Cys247 (see the main text and Supplementary Material, Fig. S5) (conditions: 15 µm of hETHE1 in 15 mm ammonium acetate buffer (pH 7.5); cone voltage for the acquisition of the spectra was 80 V). (C) MALS analysis of hETHE1 protein after purification. Peak A (∼51 560 Da) represents monomer; peak B (∼99 190 Da) the tetramer. MALS experiments were carried out by the Biophysical Services of the Biochemistry Department of Oxford University. (D) The molecular mass of hETHE1 in solution was estimated using a Sephadex G250 gel filtration column calibrated with protein standards [beta-amylase (223 kDa), albumin (66 kDa), carbonic anhydrase (29 kDa) and cytochrome C (12 kDa)].

Mentions: The hETHE1 structure reveals an αββα MBL-type fold with two central mixed β-sheets, each containing six strands, surrounded on both sides by helices (Fig. 2A and Supplementary Material, Fig. S2). In β-sheet I, β-strands 1–3 are aligned anti-parallel, with β-strands 3–6 being parallel. In β-sheet II, β-strands 7, 8, 9 and 10 are anti-parallel; β-strands 10 and 13 are parallel and β-strands 13 and 14 are anti-parallel (Supplementary Material, Fig. S2A). Secondary structural elements include: 2 β-sheets, 2 βαβα units, 8 β-hairpins, 6 β-bulges, 14 β-strands, 6 helices, 2 helix–helix interactions and 26 β-turns. Superimposition of the hETHE1 and A. thaliana ETHE1-like (PDB ID: 2GCU) structures reveals high overall fold similarity [root-mean-square deviation (RMSD) 1.43 Å over 230 Cα atoms] between the two proteins (Fig. 2B). Structure-based topology diagrams show conserved structural organization with the exception of the addition of the β11–β12 hairpin in the region linking β10 and β13 of the core fold of hETHE1 compared with the A. thaliana ETHE1 (Supplementary Material, Figs S2A and B). Topology comparisons of hETHE1 with human glyoxalase II (HAGH) and a ‘classical’ metallo-β-lactamase II (BcII) from B. cereus (Supplementary Material, Fig. S2C and D) reveal much more substantial differences in the organization of secondary structure elements. hETHE1 has been assigned as a member of the glyoxalase II family on the basis of sequence alignments (31); however, consistent with the differences in their overall folds (19), hETHE1 does not display glyoxalase II activity, when assayed using the most common glyoxalase II substrate, S-(D)-lactoylglutathione, under standard conditions (11). Enzyme-dependent oxygen consumption activity in the presence of GSSH was observed using the oxygen consumption assay as previously reported (11) (Fig. 3A). Moreover, ETHE1 does not display β-lactamase activity either using a chromogenic cephalosporin (nitrocefin), a penicillin (penicillin G) or a carbapenem (meropenem) as substrates under our standard β-lactamase assay conditions (32).Figure 2.


Crystal structure of human persulfide dioxygenase: structural basis of ethylmalonic encephalopathy.

Pettinati I, Brem J, McDonough MA, Schofield CJ - Hum. Mol. Genet. (2015)

hETHE1 activity assay and oligomerization state. (A) Oxygen consumption activity assay. hETHE1 activity was measured as percentage of oxygen consumed in the presence of GSSH. Each sample was performed in triplicate. (B) Non-denaturing electrospray soft-ionization mass spectrometry deconvoluted spectrum of purified hETHE1 protein indicates that hETHE1 is primarily dimeric. Peak A (26 170 Da) represents monomer; peak B (52370 Da) the dimer. In both monomeric and dimeric states one iron ion (+56 Da) is bound to each protomer while only in the dimer a +30 Da was observed possibly due to the oxidation state of Cys247 (see the main text and Supplementary Material, Fig. S5) (conditions: 15 µm of hETHE1 in 15 mm ammonium acetate buffer (pH 7.5); cone voltage for the acquisition of the spectra was 80 V). (C) MALS analysis of hETHE1 protein after purification. Peak A (∼51 560 Da) represents monomer; peak B (∼99 190 Da) the tetramer. MALS experiments were carried out by the Biophysical Services of the Biochemistry Department of Oxford University. (D) The molecular mass of hETHE1 in solution was estimated using a Sephadex G250 gel filtration column calibrated with protein standards [beta-amylase (223 kDa), albumin (66 kDa), carbonic anhydrase (29 kDa) and cytochrome C (12 kDa)].
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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DDV007F3: hETHE1 activity assay and oligomerization state. (A) Oxygen consumption activity assay. hETHE1 activity was measured as percentage of oxygen consumed in the presence of GSSH. Each sample was performed in triplicate. (B) Non-denaturing electrospray soft-ionization mass spectrometry deconvoluted spectrum of purified hETHE1 protein indicates that hETHE1 is primarily dimeric. Peak A (26 170 Da) represents monomer; peak B (52370 Da) the dimer. In both monomeric and dimeric states one iron ion (+56 Da) is bound to each protomer while only in the dimer a +30 Da was observed possibly due to the oxidation state of Cys247 (see the main text and Supplementary Material, Fig. S5) (conditions: 15 µm of hETHE1 in 15 mm ammonium acetate buffer (pH 7.5); cone voltage for the acquisition of the spectra was 80 V). (C) MALS analysis of hETHE1 protein after purification. Peak A (∼51 560 Da) represents monomer; peak B (∼99 190 Da) the tetramer. MALS experiments were carried out by the Biophysical Services of the Biochemistry Department of Oxford University. (D) The molecular mass of hETHE1 in solution was estimated using a Sephadex G250 gel filtration column calibrated with protein standards [beta-amylase (223 kDa), albumin (66 kDa), carbonic anhydrase (29 kDa) and cytochrome C (12 kDa)].
Mentions: The hETHE1 structure reveals an αββα MBL-type fold with two central mixed β-sheets, each containing six strands, surrounded on both sides by helices (Fig. 2A and Supplementary Material, Fig. S2). In β-sheet I, β-strands 1–3 are aligned anti-parallel, with β-strands 3–6 being parallel. In β-sheet II, β-strands 7, 8, 9 and 10 are anti-parallel; β-strands 10 and 13 are parallel and β-strands 13 and 14 are anti-parallel (Supplementary Material, Fig. S2A). Secondary structural elements include: 2 β-sheets, 2 βαβα units, 8 β-hairpins, 6 β-bulges, 14 β-strands, 6 helices, 2 helix–helix interactions and 26 β-turns. Superimposition of the hETHE1 and A. thaliana ETHE1-like (PDB ID: 2GCU) structures reveals high overall fold similarity [root-mean-square deviation (RMSD) 1.43 Å over 230 Cα atoms] between the two proteins (Fig. 2B). Structure-based topology diagrams show conserved structural organization with the exception of the addition of the β11–β12 hairpin in the region linking β10 and β13 of the core fold of hETHE1 compared with the A. thaliana ETHE1 (Supplementary Material, Figs S2A and B). Topology comparisons of hETHE1 with human glyoxalase II (HAGH) and a ‘classical’ metallo-β-lactamase II (BcII) from B. cereus (Supplementary Material, Fig. S2C and D) reveal much more substantial differences in the organization of secondary structure elements. hETHE1 has been assigned as a member of the glyoxalase II family on the basis of sequence alignments (31); however, consistent with the differences in their overall folds (19), hETHE1 does not display glyoxalase II activity, when assayed using the most common glyoxalase II substrate, S-(D)-lactoylglutathione, under standard conditions (11). Enzyme-dependent oxygen consumption activity in the presence of GSSH was observed using the oxygen consumption assay as previously reported (11) (Fig. 3A). Moreover, ETHE1 does not display β-lactamase activity either using a chromogenic cephalosporin (nitrocefin), a penicillin (penicillin G) or a carbapenem (meropenem) as substrates under our standard β-lactamase assay conditions (32).Figure 2.

Bottom Line: A channel leading to the active site is sufficiently large to accommodate a GSSH substrate.Some of the observed hETHE1 clinical mutations cluster in the active site region.The structure will serve as a basis for detailed functional and mechanistic studies on ETHE1 and will be useful in the development of selective MBL inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK.

Show MeSH
Related in: MedlinePlus