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Helicobacter pylori HP0377, a member of the Dsb family, is an untypical multifunctional CcmG that cooperates with dimeric thioldisulfide oxidase HP0231.

Roszczenko P, Grzeszczuk M, Kobierecka P, Wywial E, Urbanowicz P, Wincek P, Nowak E, Jagusztyn-Krynicka EK - BMC Microbiol. (2015)

Bottom Line: Our biochemical analysis indicates that HP0377 is a specific reductase, as it does not reduce insulin.In H. pylori HP0377 is re-reduced by CcdA (HP0265); however in E. coli it remains in the oxidized state as it does not interact with E. coli DsbD.Our in vivo work also suggests that both HP0377, which plays a role in apocytochrome reduction, and HP0378, which is involved in heme transport and its ligation into apocytochrome, provide essential functions in H. pylori.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacterial Genetics, Institute of Microbiology, Faculty of Biology, University of Warsaw, Warsaw, Poland. paula.roszczenko@gmail.com.

ABSTRACT

Background: In the genome of H. pylori 26695, 149 proteins containing the CXXC motif characteristic of thioldisulfide oxidoreductases have been identified to date. However, only two of these proteins have a thioredoxin-like fold (i.e., HP0377 and HP0231) and are periplasm-located. We have previously shown that HP0231 is a dimeric oxidoreductase that catalyzes disulfide bond formation in the periplasm. Although HP0377 was originally described as DsbC homologue, its resolved structure and location of the hp0377 gene in the genome indicate that it is a counterpart of CcmG/DsbE.

Results: The present work shows that HP0377 is present in H. pylori cells only in a reduced form and that absence of the main periplasmic oxidase HP0231 influences its redox state. Our biochemical analysis indicates that HP0377 is a specific reductase, as it does not reduce insulin. However, it possesses disulfide isomerase activity, as it catalyzes the refolding of scrambled RNase. Additionally, although its standard redox potential is -176 mV, it is the first described CcmG protein having an acidic pKa of the N-terminal cysteine of the CXXC motif, similar to E. coli DsbA or E. coli DsbC. The CcmG proteins that play a role in a cytochrome c-maturation, both in system I and system II, are kept in the reduced form by an integral membrane protein DsbD or its analogue, CcdA. In H. pylori HP0377 is re-reduced by CcdA (HP0265); however in E. coli it remains in the oxidized state as it does not interact with E. coli DsbD. Our in vivo work also suggests that both HP0377, which plays a role in apocytochrome reduction, and HP0378, which is involved in heme transport and its ligation into apocytochrome, provide essential functions in H. pylori.

Conclusions: The present data, in combination with the resolved three-dimensional structure of the HP0377, suggest that HP0377 is an unusual, multifunctional CcmG protein.

No MeSH data available.


Related in: MedlinePlus

Thiol ionization equilibrium in HP0377 followed by UV absorbance at 240 nm. Panel a: HP0377 wild type. Panel b: HP0377 C89S. Panel c: HP0377 C92S
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Fig6: Thiol ionization equilibrium in HP0377 followed by UV absorbance at 240 nm. Panel a: HP0377 wild type. Panel b: HP0377 C89S. Panel c: HP0377 C92S

Mentions: The function of thiol-oxidoreductases depends on the pKa values of their active-site Cys residues. The pKa of the HP0377 active site thiols was determined by observing the change in absorption of the thiolate anion at 240 nm as a function of pH in wt protein. The pH titration of wt HP0377 showed two transitions, one with pKa = 3.56 ± 0.11 and the other with pKa = 9.23 ± 0.21 Fig. 6a. Because these data are atypical for CcmGs, we constructed two single-Cys CXXC motif mutants of HP0377 (C89S and C92S), which allowed independent measurement of the pKa of each Cys residue. As shown in Fig. 6b, c the C89S mutant had a pKa = 3.46 ± 0.24 and the C92S mutant a pKa = 9.41 ± 0.15. These results agreed with the titrations of the wt protein. The pKa value of the solvent-exposed active site cysteine (Cys89) is about 3.5. This is relatively acidic compared with the solvent-exposed active site cysteine of the E. coli CcmG protein, pKa = 6.8 [44], or the B. subtilis ResA, pKa = 8.8 [45]; it is as acidic as E. coli DsbA, which is a known oxidant whose pKa of the solvent-exposed active site cysteine is 3.5 [38], and it is close to the pKa of EcDsbC, 4.1 ± 0.3 [46]. Thus, HP0377 is the first described CcmG protein having an acidic pKa of the N-terminal cysteine of the CXXC motif, and at the same time, it presents a low redox potential.Fig. 6


Helicobacter pylori HP0377, a member of the Dsb family, is an untypical multifunctional CcmG that cooperates with dimeric thioldisulfide oxidase HP0231.

Roszczenko P, Grzeszczuk M, Kobierecka P, Wywial E, Urbanowicz P, Wincek P, Nowak E, Jagusztyn-Krynicka EK - BMC Microbiol. (2015)

Thiol ionization equilibrium in HP0377 followed by UV absorbance at 240 nm. Panel a: HP0377 wild type. Panel b: HP0377 C89S. Panel c: HP0377 C92S
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4491210&req=5

Fig6: Thiol ionization equilibrium in HP0377 followed by UV absorbance at 240 nm. Panel a: HP0377 wild type. Panel b: HP0377 C89S. Panel c: HP0377 C92S
Mentions: The function of thiol-oxidoreductases depends on the pKa values of their active-site Cys residues. The pKa of the HP0377 active site thiols was determined by observing the change in absorption of the thiolate anion at 240 nm as a function of pH in wt protein. The pH titration of wt HP0377 showed two transitions, one with pKa = 3.56 ± 0.11 and the other with pKa = 9.23 ± 0.21 Fig. 6a. Because these data are atypical for CcmGs, we constructed two single-Cys CXXC motif mutants of HP0377 (C89S and C92S), which allowed independent measurement of the pKa of each Cys residue. As shown in Fig. 6b, c the C89S mutant had a pKa = 3.46 ± 0.24 and the C92S mutant a pKa = 9.41 ± 0.15. These results agreed with the titrations of the wt protein. The pKa value of the solvent-exposed active site cysteine (Cys89) is about 3.5. This is relatively acidic compared with the solvent-exposed active site cysteine of the E. coli CcmG protein, pKa = 6.8 [44], or the B. subtilis ResA, pKa = 8.8 [45]; it is as acidic as E. coli DsbA, which is a known oxidant whose pKa of the solvent-exposed active site cysteine is 3.5 [38], and it is close to the pKa of EcDsbC, 4.1 ± 0.3 [46]. Thus, HP0377 is the first described CcmG protein having an acidic pKa of the N-terminal cysteine of the CXXC motif, and at the same time, it presents a low redox potential.Fig. 6

Bottom Line: Our biochemical analysis indicates that HP0377 is a specific reductase, as it does not reduce insulin.In H. pylori HP0377 is re-reduced by CcdA (HP0265); however in E. coli it remains in the oxidized state as it does not interact with E. coli DsbD.Our in vivo work also suggests that both HP0377, which plays a role in apocytochrome reduction, and HP0378, which is involved in heme transport and its ligation into apocytochrome, provide essential functions in H. pylori.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacterial Genetics, Institute of Microbiology, Faculty of Biology, University of Warsaw, Warsaw, Poland. paula.roszczenko@gmail.com.

ABSTRACT

Background: In the genome of H. pylori 26695, 149 proteins containing the CXXC motif characteristic of thioldisulfide oxidoreductases have been identified to date. However, only two of these proteins have a thioredoxin-like fold (i.e., HP0377 and HP0231) and are periplasm-located. We have previously shown that HP0231 is a dimeric oxidoreductase that catalyzes disulfide bond formation in the periplasm. Although HP0377 was originally described as DsbC homologue, its resolved structure and location of the hp0377 gene in the genome indicate that it is a counterpart of CcmG/DsbE.

Results: The present work shows that HP0377 is present in H. pylori cells only in a reduced form and that absence of the main periplasmic oxidase HP0231 influences its redox state. Our biochemical analysis indicates that HP0377 is a specific reductase, as it does not reduce insulin. However, it possesses disulfide isomerase activity, as it catalyzes the refolding of scrambled RNase. Additionally, although its standard redox potential is -176 mV, it is the first described CcmG protein having an acidic pKa of the N-terminal cysteine of the CXXC motif, similar to E. coli DsbA or E. coli DsbC. The CcmG proteins that play a role in a cytochrome c-maturation, both in system I and system II, are kept in the reduced form by an integral membrane protein DsbD or its analogue, CcdA. In H. pylori HP0377 is re-reduced by CcdA (HP0265); however in E. coli it remains in the oxidized state as it does not interact with E. coli DsbD. Our in vivo work also suggests that both HP0377, which plays a role in apocytochrome reduction, and HP0378, which is involved in heme transport and its ligation into apocytochrome, provide essential functions in H. pylori.

Conclusions: The present data, in combination with the resolved three-dimensional structure of the HP0377, suggest that HP0377 is an unusual, multifunctional CcmG protein.

No MeSH data available.


Related in: MedlinePlus