Oxidative Implications of Substituting a Conserved Cysteine Residue in Sugar Beet Phytoglobin BvPgb 1.2
(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.
(Less)
- author
- Christensen, Simon LU ; Groth, Leonard LU ; Leiva-Eriksson, Nélida LU ; Nyblom, Maria and Bülow, Leif LU
- organization
- publishing date
- 2022-08
- 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
-
- pmid:36009334
- scopus:85137325861
- 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
- 2024-09-16 08:10:59
@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}}, }