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Crystal structure and biophysical properties of Bacillus subtilis BdbD: An oxidizing thiol:disulfide oxidoreductase containing a novel metal site

Crow, Allister; Lewin, Allison; Hecht, Oliver; Carlsson Möller, Mirja LU ; Moore, Geoffrey R.; Hederstedt, Lars LU and Le Brun, Nick E. (2009) In Journal of Biological Chemistry 284(35). p.23719-23733
Abstract
BdbD is a thiol: disulfide oxidoreductase (TDOR) from Bacillus subtilis that functions to introduce disulfide bonds in substrate proteins/peptides on the outside of the cytoplasmic membrane and, as such, plays a key role in disulfide bond management. Here we demonstrate that the protein is membrane-associated in B. subtilis and present the crystal structure of the soluble part of the protein lacking its membrane anchor. This reveals that BdbD is similar in structure to Escherichia coli DsbA, with a thioredoxin-like domain with an inserted helical domain. A major difference, however, is the presence in BdbD of a metal site, fully occupied by Ca2+, at an inter-domain position some 14 angstrom away from the CXXC active site. The midpoint... (More)
BdbD is a thiol: disulfide oxidoreductase (TDOR) from Bacillus subtilis that functions to introduce disulfide bonds in substrate proteins/peptides on the outside of the cytoplasmic membrane and, as such, plays a key role in disulfide bond management. Here we demonstrate that the protein is membrane-associated in B. subtilis and present the crystal structure of the soluble part of the protein lacking its membrane anchor. This reveals that BdbD is similar in structure to Escherichia coli DsbA, with a thioredoxin-like domain with an inserted helical domain. A major difference, however, is the presence in BdbD of a metal site, fully occupied by Ca2+, at an inter-domain position some 14 angstrom away from the CXXC active site. The midpoint reduction potential of soluble BdbD was determined as -75 mV versus normal hydrogen electrode, and the active site N-terminal cysteine thiol was shown to have a low pK(a), consistent with BdbD being an oxidizing TDOR. Equilibrium unfolding studies revealed that the oxidizing power of the protein is based on the instability introduced by the disulfide bond in the oxidized form. The crystal structure of Ca2+-depleted BdbD showed that the protein remained folded, with only minor conformational changes. However, the reduced form of Ca2+-depleted BdbD was significantly less stable than reduced Ca2+-containing protein, and the midpoint reduction potential was shifted by approximately -20 mV, suggesting that Ca2+ functions to boost the oxidizing power of the protein. Finally, we demonstrate that electron exchange does not occur between BdbD and B. subtilis ResA, a low potential extra-cytoplasmic TDOR. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of Biological Chemistry
volume
284
issue
35
pages
23719 - 23733
publisher
ASBMB
external identifiers
  • wos:000269180000057
  • scopus:69949125089
ISSN
1083-351X
DOI
10.1074/jbc.M109.005785
language
English
LU publication?
yes
id
4f0881df-0f1f-4dec-b3e1-7c93a9e6d1ff (old id 1477024)
date added to LUP
2009-09-24 11:00:13
date last changed
2017-08-09 15:37:38
@article{4f0881df-0f1f-4dec-b3e1-7c93a9e6d1ff,
  abstract     = {BdbD is a thiol: disulfide oxidoreductase (TDOR) from Bacillus subtilis that functions to introduce disulfide bonds in substrate proteins/peptides on the outside of the cytoplasmic membrane and, as such, plays a key role in disulfide bond management. Here we demonstrate that the protein is membrane-associated in B. subtilis and present the crystal structure of the soluble part of the protein lacking its membrane anchor. This reveals that BdbD is similar in structure to Escherichia coli DsbA, with a thioredoxin-like domain with an inserted helical domain. A major difference, however, is the presence in BdbD of a metal site, fully occupied by Ca2+, at an inter-domain position some 14 angstrom away from the CXXC active site. The midpoint reduction potential of soluble BdbD was determined as -75 mV versus normal hydrogen electrode, and the active site N-terminal cysteine thiol was shown to have a low pK(a), consistent with BdbD being an oxidizing TDOR. Equilibrium unfolding studies revealed that the oxidizing power of the protein is based on the instability introduced by the disulfide bond in the oxidized form. The crystal structure of Ca2+-depleted BdbD showed that the protein remained folded, with only minor conformational changes. However, the reduced form of Ca2+-depleted BdbD was significantly less stable than reduced Ca2+-containing protein, and the midpoint reduction potential was shifted by approximately -20 mV, suggesting that Ca2+ functions to boost the oxidizing power of the protein. Finally, we demonstrate that electron exchange does not occur between BdbD and B. subtilis ResA, a low potential extra-cytoplasmic TDOR.},
  author       = {Crow, Allister and Lewin, Allison and Hecht, Oliver and Carlsson Möller, Mirja and Moore, Geoffrey R. and Hederstedt, Lars and Le Brun, Nick E.},
  issn         = {1083-351X},
  language     = {eng},
  number       = {35},
  pages        = {23719--23733},
  publisher    = {ASBMB},
  series       = {Journal of Biological Chemistry},
  title        = {Crystal structure and biophysical properties of <em>Bacillus subtilis</em> BdbD: An oxidizing thiol:disulfide oxidoreductase containing a novel metal site},
  url          = {http://dx.doi.org/10.1074/jbc.M109.005785},
  volume       = {284},
  year         = {2009},
}