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

• 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.

Ämnesord

NATURVETENSKAP  -- Biologi -- Mikrobiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Microbiology (hsv//eng)

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

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