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Chapter 7 : Disulfide Bond Formation in the Periplasm
Category: Bacterial Pathogenesis; Microbial Genetics and Molecular Biology
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This chapter reviews the process of disulfide bond formation in the periplasm by following the life of a protein, from the “birth” of a protein at the ribosome to its entrance into the periplasm as an unfolded infant, to the oxidation of the maturing protein. In eukaryotic cells the protein disulfide isomerase (PDI) represents the counterpart of DsbA, being responsible for the formation of disulfide bonds in the endoplasmic reticulum. DsbA can act to generate disulfide bonds in proteins as nascent polypeptide chains emerge into the periplasm. Several features of disulfide bond formation discovered in recent years illustrate complexity. First, disulfide bond formation likely occurs on nascent poly-peptide chains as they are being translocated into the periplasm in bacteria and into the endoplasmic reticulum in eukaryotic cells. Second, for efficient formation of disulfide bonds, proteins require enzyme catalysts whose prototypes are DsbA in bacterial cells and protein disulfide isomerase in eukaryotic cells. Third, the formation of the correct disulfide bonds requires enzyme catalysts such as DsbC. Bioinformatic analysis conducted in the authors' lab revealed that at least 90% of the periplasmic proteins with more than one cysteine contain disulfide bonds. For those periplasmic proteins with only a single free cysteine, their crystal structures showed these cysteines to be buried within the protein structure (AlsB, AraF, FhuD, GlpQ, and Tpx). These findings are consistent with the proposal that any protein that appears in the periplasm with more than one cysteine will be acted on by DsbA.
The mechanism of disulfide bond formation. DsbA catalyzes the formation of disulfide bonds in a polypeptide with reduced cysteines. The cysteines within the Cys-X-X-Cys active site of DsbA are oxidized (S—S) and the thiol side groups of cysteine residues in the substrate are reduced (SH) ➀. Disulfide bond formation is initiated by deprotonation of a thiol group in the substrate ➁. The resulting thiolate anion can initiate a nucleophilic attack on the disulfide bond of DsbA ➂. The resolution of the mix-disulfide-bonded complex could occur by deprotonation of another thiol group ➃, which can attack the substrate-DsbA disulfide bond ➄. The result of this reaction is the oxidation of the substrate and the reduction of DsbA ➅.
Topology of DsbB. The topology of DsbB based on alkaline phos-phatase fusion studies ( Jander et al., 1994 ). The active site cysteines are shown in their oxidized state, and the putative transmem-brane domain amino acids are highlighted.
Proposed mechanism of isomerization by DsbC. For the purpose of clarity only a monomer of DsbC is shown. Reduced active DsbC recognizes misoxidized substrate ➀ and forms a mix-disulfide-bonded complex. This complex ➁ could be resolved by the reduction of the disulfide bond in the substrate, resulting in the oxidation of DsbC ➂. A secondary cycle of reduction is necessary for the substrate to be fully reduced ➄, allowing DsbA to reoxidize the substrate ➅. Alternatively, the disulfide bonds in the complex could be shuffled ➆ by the iso-merase action of DsbC, resulting in native disulfide-bonded substrate and reduced DsbC ➆.
Properties of disulfide bond-fon ming enzymes in the periplasm