The proteolytic activity of the HtrA (DegP) protein from Escherichia coli at low temperatures

Skórko-Glonek, Joanna; Sobiecka-Szkatula, Anna; Narkiewicz, Joanna; Lipińska, Barbara

doi:10.1099/mic.0.2008/020487-0

Cited by 32 publications

(28 citation statements)

References 31 publications

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“…Analysis of the initial rates indicated that the reaction was first order with respect to peptide and EcDsbA and proceeded with a second order rate constant of k ϭ 1.4 ϫ 10 6 M Ϫ1 s Ϫ1 . This is similar to the derived rate constants that have been reported previously for the reaction of DsbA enzymes with peptide and protein substrates (32)(33)(34) and indicated that the SigA peptide was efficiently oxidized by EcDsbA.…”

Section: Resultssupporting

confidence: 90%

“…Herein we report the structure of a covalent DsbA-peptide complex refined to a resolution of 1.9 Å. It has previously been found that reduced peptides and proteins are oxidized with similar kinetics (32)(33)(34), suggesting that peptides are suitable models of the reduced unfolded proteins that are the substrates of DsbA. Our structure reveals that the peptide binds to DsbA at a surface formed by residues at the interface between the ␣-helical and TRX domains and not within the hydrophobic groove that is the binding site for DsbB.…”

mentioning

confidence: 99%

“…Oxidation reaction mixtures contained EcDbsA (1-10 M) to which the SigA peptide was added over a range of concentrations (1-25 M). The initial rates of the oxidation reactions were determined from the linear portion of the fluorescence data, and these were used to determine the apparent second order rate constant for the reaction as described previously (34).…”

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confidence: 99%

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The Structure of the Bacterial Oxidoreductase Enzyme DsbA in Complex with a Peptide Reveals a Basis for Substrate Specificity in the Catalytic Cycle of DsbA Enzymes

Paxman

Borg

Horne

et al. 2009

Journal of Biological Chemistry

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Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds between pairs of cysteine residues. This is a multistep process in which the dithiol-disulfide oxidoreductase enzyme, DsbA, plays a central role. The structure of DsbA comprises an all helical domain of unknown function and a thioredoxin domain, where active site cysteines shuttle between an oxidized, substrate-bound, reduced form and a DsbB-bound form, where DsbB is a membrane protein that reoxidizes DsbA. Most DsbA enzymes interact with a wide variety of reduced substrates and show little specificity. However, a number of DsbA enzymes have now been identified that have narrow substrate repertoires and appear to interact specifically with a smaller number of substrates. The transient nature of the DsbA-substrate complex has hampered our understanding of the factors that govern the interaction of DsbA enzymes with their substrates. Here we report the crystal structure of a complex between Escherichia coli DsbA and a peptide with a sequence derived from a substrate. The binding site identified in the DsbA-peptide complex was distinct from that observed for DsbB in the DsbA-DsbB complex. The structure revealed details of the DsbA-peptide interaction and suggested a mechanism by which DsbA can simultaneously show broad specificity for substrates yet exhibit specificity for DsbB. This mode of binding was supported by solution nuclear magnetic resonance data as well as functional data, which demonstrated that the substrate specificity of DsbA could be modified via changes at the binding interface identified in the structure of the complex.

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Section: Resultssupporting

confidence: 90%

mentioning

confidence: 99%

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The Structure of the Bacterial Oxidoreductase Enzyme DsbA in Complex with a Peptide Reveals a Basis for Substrate Specificity in the Catalytic Cycle of DsbA Enzymes

Paxman

Borg

Horne

et al. 2009

Journal of Biological Chemistry

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“…The authors suggest that PepD may represent a general stress response strategy for cell wall maintenance (White et al, 2011). HtrA functions as both a chaperone and protease in E. coli and is critical for cell survival under elevated temperatures and oxidative stress conditions (Skorko-Glonek et al, 2008). With the exception of MarP in M. tuberculosis , mechanisms of serine proteases in mycobacteria remain poorly understood and understudied.…”

Section: Discussionmentioning

confidence: 99%

A Mycobacterium avium subsp. paratuberculosis Predicted Serine Protease Is Associated with Acid Stress and Intraphagosomal Survival

Kugadas

Lamont

Bannantine

et al. 2016

Front. Cell. Infect. Microbiol.

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The ability to maintain intra-cellular pH is crucial for bacteria and other microbes to survive in diverse environments, particularly those that undergo fluctuations in pH. Mechanisms of acid resistance remain poorly understood in mycobacteria. Although, studies investigating acid stress in M. tuberculosis are gaining traction, few center on Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of chronic enteritis in ruminants. We identified a MAP acid stress response network involved in macrophage infection. The central node of this network was MAP0403, a predicted serine protease that shared an 86% amino acid identity with MarP in M. tuberculosis. Previous studies confirmed MarP as a serine protease integral to maintaining intra-bacterial pH and survival in acid in vitro and in vivo. We show that MAP0403 is upregulated in infected macrophages and MAC-T cells that coincided with phagosome acidification. Treatment of mammalian cells with bafilomcyin A1, a potent inhibitor of phagosomal vATPases, diminished MAP0403 transcription. MAP0403 expression was also noted in acidic medium. A surrogate host, M. smegmatis mc2 155, was designed to express MAP0403 and when exposed to either macrophages or in vitro acid stress had increased bacterial cell viability, which corresponds to maintenance of intra-bacterial pH in acidic (pH = 5) conditions, compared to the parent strain. These data suggest that MAP0403 may be the equivalent of MarP in MAP. Future studies confirming MAP0403 as a serine protease and exploring its structure and possible substrates are warranted.

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“…Subsequent studies also suggested that DegP works as a chaperone under heat shock conditions (31) or exerts its proteolytic activity at low temperatures (33). Although the in vivo relevance of the DegP chaperone and protease activities at high and low temperatures, respectively, is unclear, these two studies certainly raise the possibility that other factors besides temperature control the protease/chaperone switch in the cell.…”

Section: Unresolved Functional Aspects Of the Degp Hexameric Formmentioning

confidence: 98%

Escherichia coli DegP: a Structure-Driven Functional Model

Iwanczyk

Jomaa

2009

J Bacteriol

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Structural biology has been extremely successful in providing functional insights for a large number of proteins and macromolecular assemblies, but in some cases, the structure has contributed a three-dimensional (3D) framework to interpret years of accumulated biochemical and genetic knowledge. In these particular systems, structural information has allowed us to learn things that would have been difficult to learn with other techniques.The periplasmic Escherichia coli DegP protease/chaperone exemplifies this scenario very well. This protein was initially identified in the 1980s (23,24,37,38), and over more than two decades, several groups characterized its activities (2,12,16,27). However, a comprehensive 3D functional model did not become apparent until the first DegP X-ray structure was revealed in 2002 (20). Following the discovery of this remarkable structure, a number of groups concentrated on testing the essentials of the functional model proposed from the crystal structure. Interestingly, this functional model was rewritten recently, after the structures of DegP in two additional oligomeric forms were resolved (13,22). Current research efforts are now concentrated on probing the new functional model and also on answering new, interesting questions posed by these structures. Therefore, DegP provides an excellent example for the structure-driven study of protein function. This minireview aims to summarize how the functional model for DegP protein has evolved as the structures of the different oligomeric forms of the protein have been elucidated.E. coli DegP (also called HtrA or protease Do) is an important periplasmic protein with the unusual property of functioning both as a protease and as a chaperone (36). Unlike the cytoplasmic compartment, the periplasm lacks ATP and does not support the function of large protein machines powered by this molecule. However, in this cellular compartment, DegP can still degrade and refold misfolded proteins in an ATPindependent manner (2). Although DegP is not an essential protein, its activity is required for bacterial survival at high temperatures (34) and under harsh environmental conditions. Consequently, its expression is upregulated by both the Cpx and E protein quality control pathways under conditions of protein-folding stress (3, 29).DegP homologs have been isolated from a variety of species, including gram-negative and -positive bacteria, plants, and mammals. All these proteins constitute the HtrA family of proteases (2). In bacteria, members of this family are key players mainly in protein quality control in the periplasmic space. In eukaryotic cells, these proteins are involved in functions as diverse as the regulation of apoptosis (1, 4, 9) and the delay of the aggregation process of intracellular amyloid peptides (19). HtrA proteins usually contain a protease domain and at least one C-terminal PDZ domain. In some cases, members of this family of proteins also include additional domains, such as transmembrane regions, located usually at the N terminus. Spec...

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The proteolytic activity of the HtrA (DegP) protein from Escherichia coli at low temperatures

Cited by 32 publications

References 31 publications

The Structure of the Bacterial Oxidoreductase Enzyme DsbA in Complex with a Peptide Reveals a Basis for Substrate Specificity in the Catalytic Cycle of DsbA Enzymes

The Structure of the Bacterial Oxidoreductase Enzyme DsbA in Complex with a Peptide Reveals a Basis for Substrate Specificity in the Catalytic Cycle of DsbA Enzymes

A Mycobacterium avium subsp. paratuberculosis Predicted Serine Protease Is Associated with Acid Stress and Intraphagosomal Survival

Escherichia coli DegP: a Structure-Driven Functional Model

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