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Open AccessArticle

A Novel Substrate-Binding Site in the X-ray Structure of an Oxidized E. coli Glyceraldehyde 3-Phosphate Dehydrogenase Elucidated by Single-Wavelength Anomalous Dispersion

1
Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Ciudad Universitaria, Coyoacán, 04510 Ciudad de México, Mexico
2
Facultad de Odontogía, Universidad Nacional Autónoma de México, Investigación Científica 1853, Ciudad Universitaria, 04360 Ciudad de México, Mexico
*
Authors to whom correspondence should be addressed.
Crystals 2019, 9(12), 622; https://doi.org/10.3390/cryst9120622
Received: 31 October 2019 / Revised: 14 November 2019 / Accepted: 22 November 2019 / Published: 26 November 2019
(This article belongs to the Special Issue Protein Crystallography)
Escherichia coli (E. coli), one of the most frequently used host for the expression of recombinant proteins, is often affected by the toxic effect of the exogenous proteins that is required to express. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a multi-functional protein that has been used as a control marker for basal function and it is known to undergo cysteine oxidation under different types of cellular stress. Here, we report the 3D structure of the endogenous GAPDH purified from stressed E. coli cells expressing a eukaryotic protein. The structure was solved at 1.64 Å using single-wavelength anomalous dispersion (SAD) phasing with a selenium-modified enzyme. Interestingly, each GAPDH monomer contains a molecule of glyceraldehyde-3 phosphate in a non-previously identified site. Furthermore, the catalytic Cys149 is covalently attached to a ~300 Da molecule, possibly glutathione. This modification alters the conformation of an adjacent alpha helix in the catalytic domain, right opposite to the NAD+ binding site. The conformation of the alpha helix is stabilized after soaking the crystals with NAD+. These results exemplify the effects that the overexpression of an exogenous protein has over the host proteins and sheds light on the structural changes that large oxidant molecules on the catalytic cysteine produce for the GAPDH enzyme. View Full-Text
Keywords: cysteine oxidation; glyceraldehyde 3-phosphate dehydrogenase; glutathione; glyceraldehyde 3-phosphate; Single-Wavelength Anomalous Dispersion (SAD); X-ray diffraction cysteine oxidation; glyceraldehyde 3-phosphate dehydrogenase; glutathione; glyceraldehyde 3-phosphate; Single-Wavelength Anomalous Dispersion (SAD); X-ray diffraction
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MDPI and ACS Style

Annia, R.-H.; Romo-Arévalo, E.; Rodríguez-Romero, A. A Novel Substrate-Binding Site in the X-ray Structure of an Oxidized E. coli Glyceraldehyde 3-Phosphate Dehydrogenase Elucidated by Single-Wavelength Anomalous Dispersion. Crystals 2019, 9, 622. https://doi.org/10.3390/cryst9120622

AMA Style

Annia R-H, Romo-Arévalo E, Rodríguez-Romero A. A Novel Substrate-Binding Site in the X-ray Structure of an Oxidized E. coli Glyceraldehyde 3-Phosphate Dehydrogenase Elucidated by Single-Wavelength Anomalous Dispersion. Crystals. 2019; 9(12):622. https://doi.org/10.3390/cryst9120622

Chicago/Turabian Style

Annia, Rodríguez-Hernández; Romo-Arévalo, Enrique; Rodríguez-Romero, Adela. 2019. "A Novel Substrate-Binding Site in the X-ray Structure of an Oxidized E. coli Glyceraldehyde 3-Phosphate Dehydrogenase Elucidated by Single-Wavelength Anomalous Dispersion" Crystals 9, no. 12: 622. https://doi.org/10.3390/cryst9120622

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