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Title: Preliminary neutron diffraction analysis of challenging human manganese superoxide dismutase crystals

Abstract

Superoxide dismutases (SODs) are enzymes that protect against oxidative stress by dismutation of superoxide into oxygen and hydrogen peroxide through cyclic reduction and oxidation of the active-site metal. The complete enzymatic mechanisms of SODs are unknown since data on the positions of hydrogen are limited. Here, we present, methods for large crystal growth and neutron data collection of human manganese SOD (MnSOD) using perdeuteration and the MaNDi beamline at Oak Ridge National Laboratory. Furthermore, The crystal from which the human MnSOD data set was obtained is the crystal with the largest unit-cell edge (240 Å) from which data have been collectedvianeutron diffraction to sufficient resolution (2.30 Å) where hydrogen positions can be observed.

Authors:
 [1];  [2]; ORCiD logo [3]; ORCiD logo [3];  [1]
  1. Nebraska Medical Center, Omaha, NE (United States). Eppley Inst. for Research in Cancer and Allied Diseases, Dept. of Biochemistry and Molecular Biology
  2. Nebraska Medical Center, Omaha, NE (United States). Eppley Inst. for Research in Cancer and Allied Diseases
  3. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biology and Soft Matter Division
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22); National Aeronautics and Space Administration (NASA); National Institutes of Health (NIH)
OSTI Identifier:
1437709
Alternate Identifier(s):
OSTI ID: 1407982
Grant/Contract Number:
AC05-00OR22725; 44-0307-1021-201; P30CA036727; 5P20RR016469
Resource Type:
Journal Article: Published Article
Journal Name:
Acta Crystallographica. Section F, Structural Biology Communications
Additional Journal Information:
Journal Volume: 73; Journal Issue: 4; Journal ID: ISSN 2053-230X
Publisher:
International Union of Crystallography
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 60 APPLIED LIFE SCIENCES; manganese superoxide dismutase; neutron diffraction; perdeuteration; human; large unit cell

Citation Formats

Azadmanesh, Jahaun, Trickel, Scott R., Weiss, Kevin L., Coates, Leighton, and Borgstahl, Gloria E. O. Preliminary neutron diffraction analysis of challenging human manganese superoxide dismutase crystals. United States: N. p., 2017. Web. doi:10.1107/S2053230X17003508.
Azadmanesh, Jahaun, Trickel, Scott R., Weiss, Kevin L., Coates, Leighton, & Borgstahl, Gloria E. O. Preliminary neutron diffraction analysis of challenging human manganese superoxide dismutase crystals. United States. doi:10.1107/S2053230X17003508.
Azadmanesh, Jahaun, Trickel, Scott R., Weiss, Kevin L., Coates, Leighton, and Borgstahl, Gloria E. O. Wed . "Preliminary neutron diffraction analysis of challenging human manganese superoxide dismutase crystals". United States. doi:10.1107/S2053230X17003508.
@article{osti_1437709,
title = {Preliminary neutron diffraction analysis of challenging human manganese superoxide dismutase crystals},
author = {Azadmanesh, Jahaun and Trickel, Scott R. and Weiss, Kevin L. and Coates, Leighton and Borgstahl, Gloria E. O.},
abstractNote = {Superoxide dismutases (SODs) are enzymes that protect against oxidative stress by dismutation of superoxide into oxygen and hydrogen peroxide through cyclic reduction and oxidation of the active-site metal. The complete enzymatic mechanisms of SODs are unknown since data on the positions of hydrogen are limited. Here, we present, methods for large crystal growth and neutron data collection of human manganese SOD (MnSOD) using perdeuteration and the MaNDi beamline at Oak Ridge National Laboratory. Furthermore, The crystal from which the human MnSOD data set was obtained is the crystal with the largest unit-cell edge (240 Å) from which data have been collectedvianeutron diffraction to sufficient resolution (2.30 Å) where hydrogen positions can be observed.},
doi = {10.1107/S2053230X17003508},
journal = {Acta Crystallographica. Section F, Structural Biology Communications},
number = 4,
volume = 73,
place = {United States},
year = {Wed Mar 29 00:00:00 EDT 2017},
month = {Wed Mar 29 00:00:00 EDT 2017}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1107/S2053230X17003508

Citation Metrics:
Cited by: 2works
Citation information provided by
Web of Science

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  • Superoxide dismutases (SODs) are enzymes that protect against oxidative stress by dismutation of superoxide into oxygen and hydrogen peroxide through cyclic reduction and oxidation of the active-site metal. The complete enzymatic mechanisms of SODs are unknown since data on the positions of hydrogen are limited. Here, we present, methods for large crystal growth and neutron data collection of human manganese SOD (MnSOD) using perdeuteration and the MaNDi beamline at Oak Ridge National Laboratory. Furthermore, The crystal from which the human MnSOD data set was obtained is the crystal with the largest unit-cell edge (240 Å) from which data have beenmore » collectedvianeutron diffraction to sufficient resolution (2.30 Å) where hydrogen positions can be observed.« less
  • Human manganese superoxide dismutase (MnSOD) is a homotetrameric enzyme which protects mitochondria against oxygen-mediated free radical damage. Within each subunit, both the N-terminal helical hairpin and C-terminal {alpha}/{beta} domains contribute ligands to the catalytic manganese site. Two identical four-helix bundles,symmetrically assembled form the N-terminal helical hairpins, form a novel tetrameric interface that stabilizes the active sites. The 2.5 {angstrom} crystallographic structure of the naturally occurring polymorphic variant Ile58Thr MnSOD reveals that the helical hairpin mutation Thr58 causes two packing defects in each of the two four-helix bundles of the tetrameric interface. Similar mutations, expected to cause packing defects in themore » Cu,ZnSOD dimer interface, are associated with the degenerative disease amyotrophic lateral sclerosis. Ile58Thr MnSOD is primarily dimeric in solution and is significantly less thermostable than the normal enzyme, with decreases of 15{degrees}C in the main melting temperature and 20{degrees}C in the heat-inactivation temperature. Consequently, this mutant MnSOD is compromised at normal body temperatures: thermal inactivation, predicted from the decrease in thermal stability, occurs with a theoretical half-life of only 3.2h at 37{degrees}C (1.4 h at 41 {degrees}C), compared with 3.1 years for native MnSOD. This prediction is supported by direct measurements: incubation at 41.7{degrees}C for 3 h has no effect on the activity of native MnSOD but completely inactivates mutant MnSOD. Rapid inactivation of Ile58Thr MnSOD at the elevated temperatures associated with fever and inflammation could provide an early advantage by killing infected cells, but also would increase superoxide-mediated oxidative damage and perhaps contribute to late-onset diseases. 63 refs., 7 figs., 2 tabs.« less
  • Recombinant clones containing the manganese superoxide dismutase (MnSOD) gene of Bacillus stearothermophilus were isolated with an oligonucleotide probe designed to match a part of the previously determined amino acid sequence. Complementation analyses, performed by introducing each plasmid into a superoxide dismutase-deficient mutant of Escherichia coli, allowed us to define the region of DNA which encodes the MnSOD structural gene and to identify a promoter region immediately upstream from the gene. These data were subsequently confirmed by DNA sequencing. Since MnSOD is normally restricted to the mitochondria in eucaryotes, we were interested (i) in determining whether B. stearothermophilus MnSOD could functionmore » in eucaryotic cytosol and (ii) in determining whether MnSOD could replace the structurally unrelated copper/zinc superoxide dismutase (Cu/ZnSOD) which is normally found there. To test this, the sequence encoding bacterial MnSOD was cloned into a yeast expression vector and subsequently introduced into a Cu/ZnSOD-deficient mutant of the yeast Saccharomyces cerevisiae. Functional expression of the protein was demonstrated, and complementation tests revealed that the protein was able to provide tolerance at wild-type levels to conditions which are normally restrictive for this mutant. Thus, in spite of the evolutionary unrelatedness of these two enzymes, Cu/ZnSOD can be functionally replaced by MnSOD in yeast cytosol.« less
  • The authors have isolated and characterized over 10,000 bp of the human EC SOD gene (SOD3 or EC 1.15.1.1) and its 5{prime}- and 3{prime}-flanking regions. Human genomic Southern blot analysis supports the existence of a single gene, without evidence for pseudogenes. The human EC SOD gene spans approximately 5900 bp. The gene can be divided into 3 exons and 2 introns. The 720-bp coding region is uninterrupted and located within exon 3. The 560 bp 5{prime} to the transcription start site were sequenced. No obvious TATA box was identified. A variety of conserved cis elements were identified by database searching.more » Exon 3 is surrounded by an Alu-J repetitive element in reverse orientation at the 5{prime} and by an Alu-Sx repetitive element in the 3{prime}-flanking DNA. The relative levels of EC SOD tissue-specific expression were determined by RNA gel blot analysis. Adult heart, placenta, pancreas, and lung had the most expression, followed by kidney, skeletal muscle, and liver. Little EC SOD message was found in the brain. A second unique mRNA, approximately 4.2 kb in length, was highly expressed in skeletal muscle. When tissue enzyme activity is compared to relative mRNA levels, there is a marked disparity in the brain, pancreas, and lung, suggesting that these tissues have enhanced affinity for circulating EC SOD or translate the EC SOD message more efficiently than other tissues. These results indicate that the EC SOD gene contains unique transcriptional regulatory elements and that its expression may be regulated at the post-transcriptional or post-translational level. The characterization of the human EC SOD gene should now allow the development of further insights into its biology and provide the basis for studies of its role in human heritable disorders. 68 refs., 5 figs., 1 tab.« less