Avoidance of the cytochrome c biogenesis system by periplasmic CXXCH motifs

2008 ◽  
Vol 36 (6) ◽  
pp. 1124-1128 ◽  
Author(s):  
Despoina A.I. Mavridou ◽  
Martin Braun ◽  
Linda Thöny-Meyer ◽  
Julie M. Stevens ◽  
Stuart J. Ferguson
Keyword(s):  
Cytochrome C ◽  
Tertiary Structure ◽  
Evolutionary Relationship ◽  
Attachment Site ◽  
Cytochrome C Maturation ◽  
E Coli ◽  
Cytochrome C Biogenesis ◽  
Periplasmic Proteins ◽  
Bona Fide

The CXXCH motif is usually recognized in the bacterial periplasm as a haem attachment site in apocytochromes c. There is evidence that the Escherichia coli Ccm (cytochrome c maturation) system recognizes little more than the CXXCH sequence. A limited number of periplasmic proteins have this motif and yet are not c-type cytochromes. To explore how unwanted haem attachment to CXXCH might be avoided, and to determine whether haem attachment to the surface of a non-cytochrome protein would be possible, we converted the active-site CXXCK motif of a thioredoxin-like protein into CXXCH, the C-terminal domain of the transmembrane oxidoreductase DsbD (cDsbD). The E. coli Ccm system was found to catalyse haem attachment to a very small percentage of the resultant protein (∼0.2%). We argue that cDsbD folds sufficiently rapidly that only a small fraction fails to avoid the Ccm system, in contrast with bona fide c-type cytochromes that only adopt their tertiary structure following haem attachment. We also demonstrate covalent haem attachment at a low level in vivo to the periplasmic disulfide isomerase DsbC, which contains a native CXXCH motif. These observations provide insight into substrate recognition by the Ccm system and expand our understanding of the requirements for covalent haem attachment to proteins. The possible evolutionary relationship between thioredoxins and c-type cytochromes is discussed.

scholarly journals The histidine of the c-type cytochrome CXXCH haem-binding motif is essential for haem attachment by the Escherichia coli cytochrome c maturation (Ccm) apparatus

2005 ◽  
Vol 389 (2) ◽  
pp. 587-592 ◽  
Author(s):  
James W. A. Allen ◽  
Nicholas Leach ◽  
Stuart J. Ferguson
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Covalent Attachment ◽  
Binding Motif ◽  
Binding Motifs ◽  
Cytochrome C Maturation ◽  
E Coli ◽  
Cytochrome B562

c-type cytochromes are characterized by covalent attachment of haem to the protein by two thioether bonds formed between the haem vinyl groups and the cysteine sulphurs in a CXXCH peptide motif. In Escherichia coli and many other Gram-negative bacteria, this post-translational haem attachment is catalysed by the Ccm (cytochrome c maturation) system. The features of the apocytochrome substrate required and recognized by the Ccm apparatus are uncertain. In the present study, we report investigations of maturation of cytochrome b562 variants containing CXXCR, CXXCK or CXXCM haem-binding motifs. None of them showed any evidence for correct maturation by the Ccm system. However, we have determined, for each variant, that the proteins (i) were expressed in large amounts, (ii) could bind haem in vivo and/or in vitro and (iii) were not degraded in the cell. Together with previous observations, these results strongly suggest that the apocytochrome substrate feature recognized by the Ccm system is simply the two cysteine residues and the histidine of the CXXCH haem-binding motif. Using the same experimental approach, we have also investigated a cytochrome b562 variant containing the special CWSCK motif that binds the active-site haem of E. coli nitrite reductase NrfA. Whereas a CWSCH analogue was matured by the Ccm apparatus in large amounts, the CWSCK form was not detectably matured either by the Ccm system or by the dedicated Nrf biogenesis proteins, implying that the substrate recognition features for haem attachment in NrfA may be more extensive than the CWSCK motif.

scholarly journals Variant c-type cytochromes as probes of the substrate specificity of the E. coli cytochrome c maturation (Ccm) apparatus

2009 ◽  
Vol 419 (1) ◽  
pp. 177-186 ◽  
Author(s):  
James W. A. Allen ◽  
Elizabeth B. Sawyer ◽  
Michael L. Ginger ◽  
Paul D. Barker ◽  
Stuart J. Ferguson
Keyword(s):  
Cytochrome C ◽  
Substrate Specificity ◽  
Cysteine Residue ◽  
Covalent Attachment ◽  
Binding Motif ◽  
Post Translational Modification ◽  
Cytochrome C Maturation ◽  
E Coli ◽  
Cytochrome B562 ◽  
Genome Analyses

c-type cytochromes are normally characterized by covalent attachment of the iron cofactor haem to protein through two thioether bonds between the vinyl groups of the haem and the thiol groups of a CXXCH (Cys–Xaa–Xaa–Cys–His) motif. In cells, the haem attachment is an enzyme-catalysed post-translational modification. We have previously shown that co-expression of a variant of Escherichia coli cytochrome b562 containing a CXXCH haem-binding motif with the E. coli Ccm (cytochrome c maturation) proteins resulted in homogeneous maturation of a correctly formed c-type cytochrome. In contrast, in the absence of the Ccm apparatus, the product holocytochrome was heterogeneous, the main species having haem inverted and attached through only one thioether bond. In the present study we use further variants of cytochrome b562 to investigate the substrate specificity of the E. coli Ccm apparatus. The system can mature c-type cytochromes with CCXXCH, CCXCH, CXCCH and CXXCHC motifs, even though these are not found naturally and the extra cysteine residue might, in principle, disrupt the biogenesis proteins which must interact intricately with disulfide-bond oxidizing and reducing proteins in the E. coli periplasm. The Ccm proteins can also attach haem to motifs of the type CXnCH where n ranges from 2 to 6. For n=3 and 4, the haem attachment was correct and homogeneous, but for higher values of n the holocytochromes displayed oxidative addition of sulfur and/or oxygen atoms associated with the covalent haem-attachment process. The implications of our observations for the haem-attachment reaction, for genome analyses and for the substrate specificity of the Ccm system, are discussed.

scholarly journals Yeast Bax Inhibitor (Bxi1p/Ybh3p) is a Calcium Channel in E. coli

2019 ◽  
Author(s):  
James Mullin ◽  
John Kalhorn ◽  
Nicholas Mello ◽  
Amanda Raffa ◽  
Alexander Strakosha ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Saccharomyces Cerevisiae ◽  
Calcium Channel ◽  
The Other ◽  
Bacterial Cells ◽  
E Coli ◽  
Founding Member ◽  
Bona Fide ◽  
Small Molecular Inhibitors

AbstractHuman Bax Inhibitor-1 (HsBI-1/TMBIM6) is the founding member of the evolutionary conserved TMBIM superfamily of proteins that share sequence homology within the transmembrane Bax inhibitor-containing motif (TMBIM). Mechanistically, BI-1/TMBIM6 and all the other mammalian TMBIM proteins appear to be involved in the maintenance of calcium homeostasis, and the crystal structure of a bacterial TMBIM protein, BsYetJ, suggests that the protein is a pH-sensitive calcium leak. The budding yeast, Saccharomyces cerevisiae, has a single TMBIM family member (YNL305C) named Bxi1p/Ybh3p. To determine the function of Bxi1p/Ybh3p, we overexpressed Bxi1p-EGFP in E. coli to determine if it is a calcium channel. We show that bacterial cells expressing Bxi1p-EGFP are more permeable to calcium than controls. Thus, our data suggests that yeast Bax inhibitor (Bxi1p) is a calcium channel in E. coli, lending support to our proposal that Bxi1p is a bona fide member of the TMBIM family of proteins. Further, we use our bacterial system to show that gadolinium is an inhibitor of Bxi1p in vivo, suggesting a path forward to identifying other small-molecular inhibitors of this clinically-important and highly conserved superfamily of proteins. Finally, parallel experiments revealed that the human Bax Inhibitor-1 (HsBI-1/TMBIM6) is also a calcium channel in bacteria that can be inhibited by gadolinium.

scholarly journals The yefM-yoeB Toxin-Antitoxin Systems of Escherichia coli and Streptococcus pneumoniae: Functional and Structural Correlation

2006 ◽  
Vol 189 (4) ◽  
pp. 1266-1278 ◽  
Author(s):  
Concha Nieto ◽  
Izhack Cherny ◽  
Seok Kooi Khoo ◽  
Mario García de Lacoba ◽  
Wai Ting Chan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Streptococcus Pneumoniae ◽  
Structural Features ◽  
E Coli ◽  
Modeled Structure ◽  
The Family ◽  
Bona Fide ◽  
K 12

ABSTRACT Toxin-antitoxin loci belonging to the yefM-yoeB family are located in the chromosome or in some plasmids of several bacteria. We cloned the yefM-yoeB locus of Streptococcus pneumoniae, and these genes encode bona fide antitoxin (YefM Spn ) and toxin (YoeB Spn ) products. We showed that overproduction of YoeB Spn is toxic to Escherichia coli cells, leading to severe inhibition of cell growth and to a reduction in cell viability; this toxicity was more pronounced in an E. coli B strain than in two E. coli K-12 strains. The YoeB Spn -mediated toxicity could be reversed by the cognate antitoxin, YefM Spn , but not by overproduction of the E. coli YefM antitoxin. The pneumococcal proteins were purified and were shown to interact with each other both in vitro and in vivo. Far-UV circular dichroism analyses indicated that the pneumococcal antitoxin was partially, but not totally, unfolded and was different than its E. coli counterpart. Molecular modeling showed that the toxins belonging to the family were homologous, whereas the antitoxins appeared to be specifically designed for each bacterial locus; thus, the toxin-antitoxin interactions were adapted to the different bacterial environmental conditions. Both structural features, folding and the molecular modeled structure, could explain the lack of cross-complementation between the pneumococcal and E. coli antitoxins.

scholarly journals Chitin, Chitin Oligosaccharide, and Chitin Disaccharide Metabolism of Escherichia coli Revisited: Reassignment of the Roles of ChiA, ChbR, ChbF, and ChbG

2021 ◽  
pp. 1-17
Author(s):  
Axel Walter ◽  
Simon Friz ◽  
Christoph Mayer
Keyword(s):  
Escherichia Coli ◽  
Catalytic Mechanism ◽  
Phosphotransferase System ◽  
E Coli ◽  
The Family ◽  
Acetyl Group ◽  
Bona Fide ◽  
Modeling Data

<i>Escherichia coli</i> is unable to grow on polymeric and oligomeric chitin, but grows on chitin disaccharide (GlcNAc-GlcNAc; <i>N,N</i>′-diacetylchitobiose) and chitin trisaccharide (GlcNAc-GlcNAc-GlcNAc; <i>N,N</i>′<i>,N</i>′′-triacetylchitotriose) via expression of the <i>chb</i> operon (<i>chbBCARFG</i>). The phosphotransferase system (PTS) transporter ChbBCA facilitates transport of both saccharides across the inner membrane and their concomitant phosphorylation at the non-reducing end, intracellularly yielding GlcNAc 6-phosphate-GlcNAc (GlcNAc6P-GlcNAc) and GlcNAc6P-GlcNAc-GlcNAc, respectively. We revisited the intracellular catabolism of the PTS products, thereby correcting the reported functions of the 6-phospho-glycosidase ChbF, the monodeacetylase ChbG, and the transcriptional regulator ChbR. Intracellular accumulation of glucosamine 6P-GlcNAc (GlcN6P-GlcNAc) and GlcN6P-GlcNAc-GlcNAc in a <i>chbF</i> mutant unraveled a role for ChbG as a monodeacetylase that removes the <i>N-</i>acetyl group at the non-reducing end. Consequently, GlcN6P- but not GlcNAc6P-containing saccharides likely function as coactivators of ChbR. Furthermore, ChbF removed the GlcN6P from the non-reducing terminus of the former saccharides, thereby degrading the inducers of the <i>chb</i> operon and facilitating growth on the saccharides. Consequently, ChbF was unable to hydrolyze GlcNAc6P-residues from the non-reducing end, contrary to previous assumptions but in agreement with structural modeling data and with the unusual catalytic mechanism of the family 4 of glycosidases, to which ChbF belongs. We also refuted the assumption that ChiA is a bifunctional endochitinase/lysozyme ChiA, and show that it is unable to degrade peptidoglycans but acts as a bona fide chitinase in vitro and in vivo, enabling growth of <i>E. coli</i> on chitin oligosaccharides when ectopically expressed. Overall, this study revises our understanding of the chitin, chitin oligosaccharide, and chitin disaccharide metabolism of <i>E. coli</i>.

What is the substrate specificity of the System I cytochrome c biogenesis apparatus?

2006 ◽  
Vol 34 (1) ◽  
pp. 150-151 ◽  
Author(s):  
J.W.A. Allen ◽  
S.J. Ferguson
Keyword(s):  
Cytochrome C ◽  
Substrate Specificity ◽  
Nitrogen Cycle ◽  
Covalent Attachment ◽  
Binding Motif ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Cytochrome C Maturation ◽  
Cytochrome C Biogenesis

c-Type cytochromes are characterized by covalent attachment of haem to protein through thioether bonds between the vinyl groups of the haem and the thiols of a CXXCH motif. Proteins of this type play crucial roles in the biochemistry of the nitrogen cycle. Many Gram-negative bacteria use the Ccm (cytochrome c maturation) proteins for the post-translational haem attachment to their c-type cytochromes; in the present paper, we discuss the substrate specificity of the Ccm apparatus. The main conclusion is that the feature recognized and required in the apocytochrome is simply the two cysteines and the histidine of the haem-binding motif.

Inhibiting Escherichia coli cytochrome c nitrite reductase: voltammetry reveals an enzyme equipped for action despite the chemical challenges it may face in vivo

2006 ◽  
Vol 34 (1) ◽  
pp. 133-135 ◽  
Author(s):  
J.D. Gwyer ◽  
D.J. Richardson ◽  
J.N. Butt
Keyword(s):  
Escherichia Coli ◽  
Cytochrome C ◽  
Nitrite Reductase ◽  
Large Family ◽  
Protein Film ◽  
E Coli ◽  
Protein Film Voltammetry ◽  
Cytochrome C Nitrite Reductase ◽  
Before And After

Escherichia coli cytochrome c nitrite reductase is one of a large family of homologous enzymes that are particularly prevalent in pathogenic enterobacteria. The enzymes are periplasmic and in vivo may find themselves challenged by molecules that could enhance or compromise their performance. In the present study, we describe protein film voltammetry in which the activity of E. coli cytochrome c nitrite reductase is challenged by the presence of a number of small molecules. These results are discussed in light of the environment(s) that the enzyme may face before and after colonization of a human host.

scholarly journals The Active-Site Cysteines of the Periplasmic Thioredoxin-Like Protein CcmG of Escherichia coli Are Important but Not Essential for Cytochrome c Maturation In Vivo

1998 ◽  
Vol 180 (7) ◽  
pp. 1947-1950 ◽  
Author(s):  
Renata A. Fabianek ◽  
Hauke Hennecke ◽  
Linda Thöny-Meyer
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Low Molecular Weight ◽  
Thiol Compounds ◽  
Cytochrome C Maturation ◽  
E Coli ◽  
The Family ◽  
Membrane Bound ◽  
Hydrophilic Domain

ABSTRACT A new member of the family of periplasmic protein thiol:disulfide oxidoreductases, CcmG (also called DsbE), was characterized with regard to its role in cytochrome c maturation in Escherichia coli. The CcmG protein was shown to be membrane bound, facing the periplasm with its C-terminal, hydrophilic domain. A chromosomal, nonpolar in-frame deletion in ccmG resulted in the complete absence of all c-type cytochromes. Replacement of either one or both of the two cysteine residues of the predicted active site in CcmG (WCPTC) led to low but detectable levels ofBradyrhizobium japonicum holocytochromec 550 expressed in E. coli. This defect, but not that of the ccmG null mutant, could be complemented by adding low-molecular-weight thiol compounds to growing cells, which is in agreement with a reducing function for CcmG.

scholarly journals Maturation of the unusual single-cysteine (XXXCH) mitochondrial c-type cytochromes found in trypanosomatids must occur through a novel biogenesis pathway

2004 ◽  
Vol 383 (3) ◽  
pp. 537-542 ◽  
Author(s):  
James W. A. ALLEN ◽  
Michael L. GINGER ◽  
Stuart J. FERGUSON
Keyword(s):  
Cytochrome C ◽  
Covalent Attachment ◽  
Binding Motif ◽  
Intermembrane Space ◽  
Cytochrome C Maturation ◽  
E Coli ◽  
Cytochromes C ◽  
Genes Encoding ◽  
Mitochondrial Intermembrane Space ◽  
Mitochondrial Cytochromes

The c-type cytochromes are characterized by the covalent attachment of haem to the polypeptide via thioether bonds formed from haem vinyl groups and, normally, the thiols of two cysteines in a CXXCH motif. Intriguingly, the mitochondrial cytochromes c and c1 from two euglenids and the Trypanosomatidae contain only a single cysteine within the haem-binding motif (XXXCH). There are three known distinct pathways by which c-type cytochromes are matured post-translationally in different organisms. The absence of genes encoding any of these c-type cytochrome biogenesis machineries is established here by analysis of six trypanosomatid genomes, and correlates with the presence of single-cysteine cytochromes c and c1. In contrast, we have identified a comprehensive catalogue of proteins required for a typical mitochondrial oxidative phosphorylation apparatus. Neither spontaneous nor catalysed maturation of the single-cysteine Trypanosoma brucei cytochrome c occurred in Escherichia coli. However, a CXXCH variant was matured by the E. coli cytochrome c maturation machinery, confirming the proposed requirement of the latter for two cysteines in the haem-binding motif and indicating that T. brucei cytochrome c can accommodate a second cysteine in a CXXCH motif. The single-cysteine haem attachment conserved in cytochromes c and c1 of the trypanosomatids is suggested to be related to their cytochrome c maturation machinery, and the environment in the mitochondrial intermembrane space. Our genomic and biochemical studies provide very persuasive evidence that the trypanosomatid mitochondrial cytochromes c are matured by a novel biogenesis system.

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