PMID:18387819

Citation	Kurz, M, Iturbe-Ormaetxe, I, Jarrott, R, Cowieson, N, Robin, G, Jones, A, King, GJ, Frei, P, Glockshuber, R, O'Neill, SL, Heras, B and Martin, JL (2008) Cloning, expression, purification and characterization of a DsbA-like protein from Wolbachia pipientis. Protein Expr. Purif. 59:266-73
Abstract	Wolbachia pipientis are obligate endosymbionts that infect a wide range of insect and other arthropod species. They act as reproductive parasites by manipulating the host reproduction machinery to enhance their own transmission. This unusual phenotype is thought to be a consequence of the actions of secreted Wolbachia proteins that are likely to contain disulfide bonds to stabilize the protein structure. In bacteria, the introduction or isomerization of disulfide bonds in proteins is catalyzed by Dsb proteins. The Wolbachia genome encodes two proteins, alpha-DsbA1 and alpha-DsbA2, that might catalyze these steps. In this work we focussed on the 234 residue protein alpha-DsbA1; the gene was cloned and expressed in Escherichia coli, the protein was purified and its identity confirmed by mass spectrometry. The sequence identity of alpha-DsbA1 for both dithiol oxidants (E. coli DsbA, 12%) and disulfide isomerases (E. coli DsbC, 14%) is similar. We therefore sought to establish whether alpha-DsbA1 is an oxidant or an isomerase based on functional activity. The purified alpha-DsbA1 was active in an oxidoreductase assay but had little isomerase activity, indicating that alpha-DsbA1 is DsbA-like rather than DsbC-like. This work represents the first successful example of the characterization of a recombinant Wolbachia protein. Purified alpha-DsbA1 will now be used in further functional studies to identify protein substrates that could help explain the molecular basis for the unusual Wolbachia phenotypes, and in structural studies to explore its relationship to other disulfide oxidoreductase proteins.
Links	PubMed Online version:10.1016/j.pep.2008.02.008
Keywords	Amino Acid Sequence; Bacterial Proteins/biosynthesis; Bacterial Proteins/chemistry; Bacterial Proteins/isolation & purification; Cloning, Molecular; Escherichia coli/genetics; Molecular Sequence Data; Protein Disulfide-Isomerases/biosynthesis; Protein Disulfide-Isomerases/chemistry; Protein Disulfide-Isomerases/isolation & purification; Sequence Alignment; Wolbachia/enzymology; Wolbachia/genetics
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
WOLPM:Q73GA4		GO:0019153: protein-disulfide reductase (glutathione) activity	ECO:0000314:		F		The protein identifier is not present in the paper, but the locus number WD_1055 and whole genome accession number AE017196 are provided in the beginning of the material and method section. Genbank search finds the protein protein_id="AAS14712.1"/note="Identified by similarity to GP:15155781" Fig.5 "showed that a-DsbA1 exhibits oxidoreductase activity in the insu- lin reductase activity, at about the same level of activity as EcDsbA."	complete CACAO 8137
WOLPM:Q73GA4		GO:0003756: protein disulfide isomerase activity	ECO:0000314:		F		The protein identifier is not present in the paper, but the locus number WD_1055 and whole genome accession number AE017196 are provided in the beginning of the material and method section. Genbank search finds the protein protein_id="AAS14712.1"/note="Identified by similarity to GP:15155781" Fig.6 "a-DsbA1 has an activity similar to that of EcDsbA in the ScRNaseA isomerase assay."	complete CACAO 8138
WOLPM:Q73GA4	enables	GO:0019153: protein-disulfide reductase (glutathione) activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
WOLPM:Q73GA4	enables	GO:0003756: protein disulfide isomerase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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