Impact of selected amino acids of HP0377 (Helicobacter pylori thiol oxidoreductase) on its functioning as a CcmG (cytochrome c maturation) protein and Dsb (disulfide bond) isomerase

Abstract Background Phytases are widely used commercially as dietary supplements for swine and poultry to increase the digestibility of phytic acid. Enzyme development has focused on increasing thermostability to withstand the high temperatures during industrial steam pelleting. Increasing thermostability often reduces activity at gut temperatures and there remains a demand for improved phyases for a growing market. Results In this work, we present a thermostable variant of the E. coli AppA phytase, ApV1, that contains an extra non-consecutive disulfide bond. Detailed biochemical characterisation of ApV1 showed similar activity to the wild type, with no statistical differences in kcat and KM for phytic acid or in the pH and temperature activity optima. Yet, it retained approximately 50% activity after incubations for 20 min at 65, 75 and 85 °C compared to almost full inactivation of the wild-type enzyme. Production of ApV1 in Pichia pastoris (Komagataella phaffi) was much lower than the wild-type enzyme due to the presence of the extra non-consecutive disulfide bond. Production bottlenecks were explored using bidirectional promoters for co-expression of folding chaperones. Co-expression of protein disulfide bond isomerase (Pdi) increased production of ApV1 by ~ 12-fold compared to expression without this folding catalyst and restored yields to similar levels seen with the wild-type enzyme. Conclusions Overall, the results show that protein engineering for enhanced enzymatic properties like thermostability may result in folding complexity and decreased production in microbial systems. Hence parallel development of improved production strains is imperative to achieve the desirable levels of recombinant protein for industrial processes.

Download Full-text

The critical role of cytochrome c maturation (CCM) system in the tolerance of Xanthomonas campestris pv. campestris to phenazines

Pesticide Biochemistry and Physiology ◽

10.1016/j.pestbp.2019.02.003 ◽

2019 ◽

Vol 156 ◽

pp. 63-71 ◽

Cited By ~ 2

Author(s):

Jian Wu ◽

Xiayan Pan ◽

Shu Xu ◽

Yabing Duan ◽

Jianying Luo ◽

...

Keyword(s):

Cytochrome C ◽

Xanthomonas Campestris ◽

Critical Role ◽

Cytochrome C Maturation ◽

Xanthomonas Campestris Pv Campestris

Download Full-text

The amino acid sequence of cytochromes c-551 from three species of Pseudomonas

Biochemical Journal ◽

10.1042/bj1310485 ◽

1973 ◽

Vol 131 (3) ◽

pp. 485-498 ◽

Cited By ~ 113

Author(s):

R. P. Ambler ◽

Margaret Wynn

Keyword(s):

Amino Acids ◽

Pseudomonas Aeruginosa ◽

Amino Acid ◽

Cytochrome C ◽

Amino Acid Sequence ◽

Amino Acid Sequences ◽

Mitochondrial Cytochrome ◽

Cytochromes C ◽

Lending Library ◽

Single Peptide

The amino acid sequences of the cytochromes c-551 from three species of Pseudomonas have been determined. Each resembles the protein from Pseudomonas strain P6009 (now known to be Pseudomonas aeruginosa, not Pseudomonas fluorescens) in containing 82 amino acids in a single peptide chain, with a haem group covalently attached to cysteine residues 12 and 15. In all four sequences 43 residues are identical. Although by bacteriological criteria the organisms are closely related, the differences between pairs of sequences range from 22% to 39%. These values should be compared with the differences in the sequence of mitochondrial cytochrome c between mammals and amphibians (about 18%) or between mammals and insects (about 33%). Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication SUP 50015 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973), 131, 5.

Download Full-text

Characterization of Soluble, Disulfide Bond-Stabilized, Prokaryotically Expressed Human Thyrotropin Receptor Ectodomain*

Endocrinology ◽

10.1210/endo.138.2.4920 ◽

1997 ◽

Vol 138 (2) ◽

pp. 588-593 ◽

Cited By ~ 8

Author(s):

Y. Bobovnikova ◽

P. N. Graves ◽

H. Vlase ◽

T. F. Davies

Keyword(s):

Amino Acids ◽

Disulfide Bond ◽

Disulfide Bonds ◽

Thioredoxin Reductase ◽

Biologically Active ◽

Receptor Protein ◽

Enzyme Linked Immunosorbent Assay ◽

Fusion Partner ◽

E Coli ◽

Recombinant Receptor

Abstract To study the interaction of TSH receptor (TSHR) autoantibodies with receptor protein, it is necessary first to express the receptor in the proper conformation including the formation of correct disulfide bridges. However, the reducing environment of the Escherichia coli (E. coli) cytoplasm prevents the generation of protein disulfide bonds and limits the solubility and immunoreactivity of recombinant human TSHR (hTSHR) products. To circumvent these limitations, hTSHR complementary DNA encoding the extracellular domain (hTSHR-ecd; amino acids 21–415) was inserted into the vector pGEX-2TK by directional cloning and used to transform the thioredoxin reductase mutant strain of E. coli (Ad494), which allowed formation of disulfide bonds in the cytoplasm. After induction, the expressed soluble hTSHR-ecd fusion protein was detected by Western blot analysis using a monoclonal antibody directed against hTSHR amino acids 21–35. This showed that over 50% of the expressed hTSHR-ecd was soluble in contrast to expression in a wild-type E. coli (strain αF′), where the majority of the recombinant receptor was insoluble. The soluble recombinant receptor was affinity purified and characterized. Under nonreducing SDS-PAGE conditions, the soluble hTSHR-ecd migrated as refolded, disulfide bond-stabilized, multimeric species, whose formation was independent of fusion partner protein. This product was found to be biologically active as evidenced by the inhibition of the binding of 125I-TSH to the full-length hTSHR expressed in transfected CHO cells and was used to develop a competitive capture enzyme-linked immunosorbent assay for mapping of hTSHR antibody epitopes. Hence, hTSHR-ecd produced in bacteria with a thioredoxin reductase mutation was found to be highly soluble and biologically relevant.

Download Full-text

The role of disulfide bond isomerase A (DsbA) ofEscherichia coliO157:H7 in biofilm formation and virulence

FEMS Microbiology Letters ◽

10.1111/j.1574-6968.2007.00993.x ◽

2008 ◽

Vol 278 (2) ◽

pp. 213-222 ◽

Cited By ~ 26

Author(s):

Yunho Lee ◽

Younghoon Kim ◽

Sujin Yeom ◽

Saehun Kim ◽

Sungsu Park ◽

...

Keyword(s):

Disulfide Bond ◽

Biofilm Formation ◽

Disulfide Bond Isomerase

Download Full-text

An In Vitro Model to Investigate the Role of Helicobacter pylori in Type 2 Diabetes, Obesity, Alzheimer’s Disease and Cardiometabolic Disease

International Journal of Molecular Sciences ◽

10.3390/ijms21218369 ◽

2020 ◽

Vol 21 (21) ◽

pp. 8369

Author(s):

Paola Cuomo ◽

Marina Papaianni ◽

Clementina Sansone ◽

Antonio Iannelli ◽

Domenico Iannelli ◽

...

Keyword(s):

Type 2 Diabetes ◽

Amino Acids ◽

Helicobacter Pylori ◽

Bacterial Colonization ◽

Mammalian Target Of Rapamycin ◽

Disease Model ◽

Cardiometabolic Disease ◽

Isoleucine Leucine

Helicobacter pylori (Hp) is a Gram-negative bacterium colonizing the human stomach. Nuclear Magnetic Resonance (NMR) analysis of intracellular human gastric carcinoma cells (MKN-28) incubated with the Hp cell filtrate (Hpcf) displays high levels of amino acids, including the branched chain amino acids (BCAA) isoleucine, leucine, and valine. Polymerase chain reaction (PCR) Array Technology shows upregulation of mammalian Target Of Rapamycin Complex 1 (mTORC1), inflammation, and mitochondrial dysfunction. The review of literature indicates that these traits are common to type 2 diabetes, obesity, Alzheimer’s diseases, and cardiometabolic disease. Here, we demonstrate how Hp may modulate these traits. Hp induces high levels of amino acids, which, in turn, activate mTORC1, which is the complex regulating the metabolism of the host. A high level of BCAA and upregulation of mTORC1 are, thus, directly regulated by Hp. Furthermore, Hp modulates inflammation, which is functional to the persistence of chronic infection and the asymptomatic state of the host. Finally, in order to induce autophagy and sustain bacterial colonization of gastric mucosa, the Hp toxin VacA localizes within mitochondria, causing fragmentation of these organelles, depletion of ATP, and oxidative stress. In conclusion, our in vitro disease model replicates the main traits common to the above four diseases and shows how Hp may potentially manipulate them.

Download Full-text