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Oxidative Implications of Substituting a Conserved Cysteine Residue in Sugar Beet Phytoglobin BvPgb 1.2

Christensen, Simon LU orcid ; Groth, Leonard LU ; Leiva-Eriksson, Nélida LU orcid ; Nyblom, Maria and Bülow, Leif LU (2022) In Antioxidants 11(8).
Abstract

Phytoglobins (Pgbs) are plant-originating heme proteins of the globin superfamily with varying degrees of hexacoordination. Pgbs have a conserved cysteine residue, the role of which is poorly understood. In this paper, we investigated the functional and structural role of cysteine in BvPgb1.2, a Class 1 Pgb from sugar beet (Beta vulgaris), by constructing an alanine-substituted mutant (Cys86Ala). The substitution had little impact on structure, dimerization, and heme loss as determined by X-ray crystallography, size-exclusion chromatography, and an apomyoglobin-based heme-loss assay, respectively. The substitution significantly affected other important biochemical properties. The autoxidation rate increased 16.7- and 14.4-fold for the... (More)

Phytoglobins (Pgbs) are plant-originating heme proteins of the globin superfamily with varying degrees of hexacoordination. Pgbs have a conserved cysteine residue, the role of which is poorly understood. In this paper, we investigated the functional and structural role of cysteine in BvPgb1.2, a Class 1 Pgb from sugar beet (Beta vulgaris), by constructing an alanine-substituted mutant (Cys86Ala). The substitution had little impact on structure, dimerization, and heme loss as determined by X-ray crystallography, size-exclusion chromatography, and an apomyoglobin-based heme-loss assay, respectively. The substitution significantly affected other important biochemical properties. The autoxidation rate increased 16.7- and 14.4-fold for the mutant versus the native protein at 25 °C and 37 °C, respectively. Thermal stability similarly increased for the mutant by ~2.5 °C as measured by nano-differential scanning fluorimetry. Monitoring peroxidase activity over 7 days showed a 60% activity decrease in the native protein, from 33.7 to 20.2 U/mg protein. When comparing the two proteins, the mutant displayed a remarkable enzymatic stability as activity remained relatively constant throughout, albeit at a lower level, ~12 U/mg protein. This suggests that cysteine plays an important role in BvPgb1.2 function and stability, despite having seemingly little effect on its tertiary and quaternary structure.

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author
; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
autoxidation, crystallization, heme loss, hexacoordination, peroxidase activity, phytoglobin, thermal stability
in
Antioxidants
volume
11
issue
8
article number
1615
publisher
MDPI AG
external identifiers
  • scopus:85137325861
  • pmid:36009334
ISSN
2076-3921
DOI
10.3390/antiox11081615
language
English
LU publication?
yes
id
023a400c-8b6b-4763-866e-50b3cfd16132
date added to LUP
2022-11-29 14:40:28
date last changed
2024-06-23 23:51:43
@article{023a400c-8b6b-4763-866e-50b3cfd16132,
  abstract     = {{<p>Phytoglobins (Pgbs) are plant-originating heme proteins of the globin superfamily with varying degrees of hexacoordination. Pgbs have a conserved cysteine residue, the role of which is poorly understood. In this paper, we investigated the functional and structural role of cysteine in BvPgb1.2, a Class 1 Pgb from sugar beet (Beta vulgaris), by constructing an alanine-substituted mutant (Cys86Ala). The substitution had little impact on structure, dimerization, and heme loss as determined by X-ray crystallography, size-exclusion chromatography, and an apomyoglobin-based heme-loss assay, respectively. The substitution significantly affected other important biochemical properties. The autoxidation rate increased 16.7- and 14.4-fold for the mutant versus the native protein at 25 °C and 37 °C, respectively. Thermal stability similarly increased for the mutant by ~2.5 °C as measured by nano-differential scanning fluorimetry. Monitoring peroxidase activity over 7 days showed a 60% activity decrease in the native protein, from 33.7 to 20.2 U/mg protein. When comparing the two proteins, the mutant displayed a remarkable enzymatic stability as activity remained relatively constant throughout, albeit at a lower level, ~12 U/mg protein. This suggests that cysteine plays an important role in BvPgb1.2 function and stability, despite having seemingly little effect on its tertiary and quaternary structure.</p>}},
  author       = {{Christensen, Simon and Groth, Leonard and Leiva-Eriksson, Nélida and Nyblom, Maria and Bülow, Leif}},
  issn         = {{2076-3921}},
  keywords     = {{autoxidation; crystallization; heme loss; hexacoordination; peroxidase activity; phytoglobin; thermal stability}},
  language     = {{eng}},
  number       = {{8}},
  publisher    = {{MDPI AG}},
  series       = {{Antioxidants}},
  title        = {{Oxidative Implications of Substituting a Conserved Cysteine Residue in Sugar Beet Phytoglobin BvPgb 1.2}},
  url          = {{http://dx.doi.org/10.3390/antiox11081615}},
  doi          = {{10.3390/antiox11081615}},
  volume       = {{11}},
  year         = {{2022}},
}