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5.A.1 The Disulfide Bond Oxidoreductase D (DsbD) Family

The best characterized member of the DsbD family is DsbD of E. coli (Katzen and Beckwith, 2000; Krupp et al., 2001). The DsbD protein is membrane-embedded with a putative N-terminal TMS plus 8 additional TMSs. The smallest homologues (190 aas with 6 putative TMSs) are found in archaea while the largest are found in both Gram-negative bacteria (758 aas with 9 putative TMSs) and Gram-positive bacteria (695 aas with 6 putative TMSs). In the E. coli DsbD system, electrons are transferred from NADPH in the cytoplasm to periplasmic dithiol/disulfide-containing proteins via an electron transfer chain that sequentially involves NADPH, thioredoxin reductase (TrxB), present in the cytoplasm, thioredoxin (TrxA), also in the cytoplasm, DsbD, the integral membrane constituent of the system, and the periplasmic electron acceptors, DsbC, DsbE (CcmG) and DsbG. All of these last three proteins can donate electrons to oxidized disulfide-containing proteins in the periplasm of a Gram-negative bacterium or presumably in the external milieu of a Gram-positive bacterium or an archaeon. Thus, the pathway is:

NADPH → TrxB → TrxA → DsbD → DsbC, DsbE, or DsbG → proteins.

Homologues include: (1) several thiol-disufide exchange proteins, (2) the cytochrome c-type biogenesis proteins, CcdA of Paracoccus pantotrophus and Bacillus subtilis (Bardischewsky and Friedrich, 2001; Le Brun et al., 2000), (3) the methylamine utilization proteins, MauF of Paracoccus denitrificans and P. versutus (Chistoserdov et al., 1992; Van Spanning et al., 1994), (4) the mercury resistance proteins (possibly Hg2+ transporters) of Mycobacterium tuberculosis and Streptomyces lividans (Brunker et al., 1996; Sedlmeier and Altenbuchner, 1992), (5) suppressors of copper sensitivity (copper tolerance proteins) of Salmonella typhimurium and Vibrio cholerae (Choudhury and Kumar, 1996; Gupta et al., 1997), (6) coomponents of peroxide reduction pathways, and (7) components of sulfenic acid reductases.  

DsbD contains three domains, each containing two reactive cysteines. One membrane-embedded domain, DsbDγ, transfers electrons from thioredoxin to the carboxy-terminal thioredoxin-like periplasmic domain DsbDγ. Alanines were substituted for each of 19 conserved amino acid residues. 11 mutants caused defects in DsbC reduction. To analyze the redox state of each DsbD domain, a thrombin-cleavable DsbD (DsbDTH) was constructed from which all three domains as separate polypeptide chains were generated. Mutants with strong defects included one mutant class that could not receive electrons from cytoplasmic thioredoxin, resulting in a DsbD that has all six of its cysteines disulfide bonded. One mutant class could not transfer electrons from DsbDβ to DsbDγ (Cho and Beckwith, 2006).

DsbD contains three cysteine pairs that undergo reversible disulfide rearrangements (Krupps et al., 2001). TrxA donates electrons to the transmembrane cysteines C163 (C3) and C285 (C5) in putative TMSs 1 and 4 in the DsbD model proposed by Katzen and Beckwith (2000). This dithiol then donates electrons to the periplasmic C-terminal thioredoxin motif (CXXC) of DsbD, thereby reducing C461 and C464 (C6 and C7, respectively). This dithiol pair attacks the periplasmic N-terminal disulfide bridge at C103 and C109 (C1 and C2, respectively) which transfers electrons to DsbC and other protein electron acceptors as noted above. The substrates of DsbD include protein disulfide isomerases and a protein involved in cytochrome c assembly (Porat et al. 2004).

DsbD catalyses an essentially irreversible reaction due to the fact that electrons flow down their electrochemical gradient from inside the cell (negative inside) to outside the cell (positive outside). In order to reverse the reaction, electrons are transferred from dithiol proteins in the periplasm to an electron acceptor in the cytoplasm as follows:

reduced proteinperiplasm → DsbAperiplasm → DsbBmembrane → quinonesmembrane → reductasemembrane
→ terminal electron acceptorcytoplasm (e.g., O2, NO3- or fumarate).

DsbB contains 4 essential cysteine residues, reversibly forming two disulfide bonds. Although DsbA displays no proofreading activity for repair of wrongly paired disulfides, this activity is displayed by DsbC, DsbE and DsbG (Krupp et al., 2001). Therefore, the two transmembrane pathways involving DsbD and DsbB together catalyze extracellular disulfide reduction (DsbD) and oxidation (DsbB) in a superficially reversible process that allows dithiol/disulfide exchange.

A class of DsbD proteins, named ScsB, are found in proteobacteria and Chlamydia. ScsB has a domain organization similar to that of DsbD, but its amino-terminal domain differs. In DsbD, this domain directly interacts with substrates to reduce them, which suggests that ScsB acts on a different array of substrates. Using Caulobacter crescentus as a model organism, Cho et al. 2012 searched for the substrates of ScsB. ScsB provides electrons to a peroxide reduction pathway in the bacterial cell envelope. The reduction pathway comprises a thioredoxin-like protein, TlpA, and a peroxiredoxin, PprX. PprX is a thiol-dependent peroxidase that efficiently reduces both hydrogen peroxide and organic peroxides. Additional proteins that depend on ScsB for reduction include a peroxiredoxin-like protein, PrxL, and a novel protein disulfide isomerase, ScsC.  Thus, the array of proteins involved in reductive pathways in the oxidative cell envelope is broad. 

The best-known TM reductase is E. coli DsbD, which has a TM domain (DsbDβ) and two periplasmic domains (DsbDα and DsbDγ), each containing a redox-active cysteine pair.  Electrons are transferred from cytoplasmic thioredoxin A (TrxA) to DsbDβ, relayed across the membrane and through its periplasmic domains, and transferred to various periplasmic targets to maintain their reductive activity. Redox activities (and their associated proteins) include protein disulfide isomerization (DsbC), cytochrome c maturation (CcmG) and defense against oxidative stress (DsbG) (Williamson et al. 2015).

CcdA is a minimal homolog of DsbDβ that has six TMSs instead of eight as in DsbDβ and can also reduce CcmG. Another DsbD homolog, ScsB, found in Salmonella typhimurium, also lacks the last two TMSs but maintains similar periplasmic domains and functionality, thus suggesting that the last two TMSs are dispensable. Separate DsbDα homologs exist in many bacteria with CcdA only. It has been proposed that CcdA has evolved into DsbD through fusion of the two periplasmic proteins to the TM domain, thus allowing greater electron-transfer efficiency (Williamson et al. 2015).

The transporter-like architecture of DsbDβ has been suggested from functional mutagenesis and biochemical studies probing solvent accessibility. The two functional cysteines of DsbDβ are on predicted TMSs 1 and 4. The sequence surrounding the cysteines is highly conserved, particularly the proline residues flanking each cysteine. Each proline in the PCX(2-3)P active sites is important for cysteine accessibility and redox activity. DsbDβ has an inverted pseudosymmetry, thus allowing access to the functional cysteines from both sides of the membrane, which allows the two cysteines to cycle through oxidized and reduced states and thus pass reducing power into the periplasm (Williamson et al. 2015).

The overall vectorial electron transfer reaction catalyzed by DsbD is:

2 e-cytoplasm → 2 e-periplasm

This family belongs to the: LysE Superfamily.

References associated with 5.A.1 family:

Appia-Ayme, C. and B.C. Berks. (2002). SoxV, an orthologue of the CcdA disulfide transporter, is involved in thiosulfate oxidation in Rhodovulum sulfidophilum and reduces the periplasmic thioredoxin SoxW. Biochem. Biophys. Res. Commun. 296: 737-741. 12176044
Bardischewsky, F. and C.G. Friedrich. (2001). Identification of CcdA in Paracoccus pantotrophus GB17: disruption of ccdA causes complete deficiency in c-type cytochromes. J. Bacteriol. 183: 257-263. 11114924
Brunker, P., D. Rother, R. Sedlmeier, J. Klein, R. Mattes, and J. Altenbuchner. (1996). Regulation of the operon responsible for broad-spectrum mercury resistance in Streptomyces lividans 1326. Mol. Genet. 251: 307-315. 8676873
Chistoserdov, A.Y., J. Boyd, F.S. Mathews, and M.E. Lidstrom. (1992). The genetic organization of the mau gene cluster of the facultative autotroph Paracoccus denitrificans. Biochem. Biophys. Res. Commun. 184: 1181-1189. 1590782
Cho, S.H. and J.F. Collet. (2013). Many roles of the bacterial envelope reducing pathways. Antioxid Redox Signal 18: 1690-1698. 23025488
Cho, S.H., and J. Beckwith. (2006). Mutations of the Membrane-Bound Disulfide Reductase DsbD That Block Electron Transfer Steps from Cytoplasm to Periplasm in Escherichia coli. J. Bacteriol. 188: 5066-5076. 16816179
Cho, S.H., D. Parsonage, C. Thurston, R.J. Dutton, L.B. Poole, J.F. Collet, and J. Beckwith. (2012). A new family of membrane electron transporters and its substrates, including a new cell envelope peroxiredoxin, reveal a broadened reductive capacity of the oxidative bacterial cell envelope. MBio 3:. 22493033
Choudhury, P. and R. Kumar. (1996). Association of metal tolerance with multiple antibiotic resistance of enteropathogenic organisms isolated from coastal region of deltaic Sunderbans. Indian J. Med. Res. 104: 148-151. 8783519
Collet, J.-F. and J.C.A. Bardwell. (2002). Oxidative protein folding in bacteria. Mol. Microbiol. 44: 1-8. 11967064
Goldstone, D.C., P. Metcalf, and E.N. Baker. (2016). Structure of the ectodomain of the electron transporter Rv2874 from Mycobacterium tuberculosis reveals a thioredoxin-like domain combined with a carbohydrate-binding module. Acta Crystallogr D Struct Biol 72: 40-48. 26894533
Gupta, S.D., H.C. Wu, and P.D. Rick. (1997). A Salmonella typhimurium genetic locus which confers copper tolerance on copper-sensitive mutants of Escherichia coli. J. Bacteriol. 179: 4977-4984. 9260936
Katzen, F. and J. Beckwith. (2000). Transmembrane electron transfer by the membrane protein DsbD occurs via a disulfide bond cascade. Cell 103: 769-779. 11114333
Kimball, R.A., L. Martin, and M.H. Saier, Jr. (2003). Reversing transmembrane electron flow: The DsbD and DsbB protein families. J. Mol. Microbiol. Biotechnol. 5: 133-149. 12766342
Krupp, R., C. Chan, and D. Missiakas. (2001). DsbD-catalyzed transport of electrons across the membrane of Escherichia coli. J. Biol. Chem. 276: 3696-3701. 11085993
Le Brun, N.E., J. Bengtsson, and L. Hederstedt. (2000). Genes required for cytochrome c synthesis in Bacillus subtilis. Mol. Microbiol. 36: 638-650. 10844653
Porat, A., S.H. Cho, and J. Beckwith. (2004). The unusual transmembrane electron transporter DsbD and its homologues: a bacterial family of disulfide reductases. Res. Microbiol. 155: 617-622. 15380548
Rozhkova, A., C.U. Stirnimann, P. Frei, U. Grauschopf, R. Brunisholz, M.G. Grütter, G. Capitani, and R. Glockshuber. (2004). Structural basis and kinetics of inter- and intramolecular disulfide exchange in the redox catalyst DsbD. EMBO. J. 23: 1709-1719. 15057279
Sedlmeier, R. and J. Altenbuchner. (1992). Cloning and DNA sequence analysis of the mercury resistance genes of Streptomyces lividans. Mol. Gen. Genet. 236: 76-85. 1494353
Stirnimann, C.U., A. Rozhkova, U. Grauschopf, R.A. Böckmann, R. Glockshuber, G. Capitani, and M.G. Grütter. (2006). High-resolution structures of Escherichia coli cDsbD in different redox states: A combined crystallographic, biochemical and computational study. J. Mol. Biol. 358: 829-845. 16545842
Van Spanning, R.J., C.J. van der Palen, D.J. Slotboom, W.N. Reijnders, A.H. Stouthamer, and J.A. Duine. (1994). Expression of the mau genes involved in methylamine metabolism in Paracoccus denitrificans is under control of a LysR-type transcriptional activator. Eur. J. Biochem. 226: 201-210. 7957249
Williamson, J.A., S.H. Cho, J. Ye, J.F. Collet, J.R. Beckwith, and J.J. Chou. (2015). Structure and multistate function of the transmembrane electron transporter CcdA. Nat Struct Mol Biol 22: 809-814. 26389738
Zhou, Y. and J.H. Bushweller. (2018). Solution structure and elevator mechanism of the membrane electron transporter CcdA. Nat Struct Mol Biol 25: 163-169. 29379172