The effect of the OmpT protease on excision repair in UV-irradiated Escherichia coli

Sedliaková, Milena; Mašek, František; Slezáriková, V.; Piršel, Miroslav

doi:10.1016/s1011-1344(97)00111-5

Cited by 8 publications

(3 citation statements)

References 15 publications

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“…Escherichia coli OmpT (EC 3.4.21.87) is a 33.5 kDa outer membrane protease that cleaves peptides and proteins preferentially between two consecutive basic amino acids [1–5]. The enzyme has been suggested to be involved in urinary tract disease [6], in DNA excision repair [7] and in the breakdown of antimicrobial peptides [8], but its actual biological function remains to be elucidated. OmpT is a member of the omptin family that includes the proteases PgtE of Salmonella typhimurium (mature part 49% identical to OmpT) [9], Pla of Yersinia pestis (50% identical) [10], OmpP of E. coli (72% identical) [11] and SopA of Shigella flexneri (60% identical) [12].…”

Section: Introductionmentioning

confidence: 99%

Identification of essential acidic residues of outer membrane protease OmpT supports a novel active site

et al. 2001

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Escherichia coli outer membrane protease OmpT has previously been classified as a serine protease with Ser 99 and His 212 as active site residues. The recently solved X-ray structure of the enzyme was inconsistent with this classification, and the involvement of a nucleophilic water molecule was proposed. Here, we substituted all conserved aspartate and glutamate residues by alanines and measured the residual enzymatic activities of the variants. Our results support the involvement of a nucleophilic water molecule that is activated by the Asp 210 / His 212 catalytic dyad. Activity is also strongly dependent on Asp 83 and Asp 85 . Both may function in binding of the water molecule and/or oxyanion stabilization. The proposed mechanism implies a novel proteolytic catalytic site. ß

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Section: Introductionmentioning

confidence: 99%

Identification of essential acidic residues of outer membrane protease OmpT supports a novel active site

et al. 2001

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“…The enzyme cleaves peptides and proteins preferentially between two consecutive basic amino acids [1]. OmpT has been suggested to be involved in urinary tract disease [3], in DNA excision repair [4] and in the breakdown of antimicrobial peptides [5], but the exact biological function remains to be elucidated.…”

Section: Introductionmentioning

confidence: 99%

Identification of active site serine and histidine residues in Escherichia coli outer membrane protease OmpT

2000

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“…However, the recently solved X-ray structure of OmpT [10] and mutagenesis studies of conserved acidic residues [11] indicate that OmpT might be a novel protease with an Asp/His catalytic dyad. The enzyme has been suggested to be involved in urinary tract disease [12], in DNA excision repair [13] and in the breakdown of antimicrobial peptides [14], but the exact biological function remains unclear. We previously developed an in vitro folding procedure to facilitate large-scale purification of OmpT from inclusion bodies [5].…”

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confidence: 99%

Lipopolysaccharide regions involved in the activation of Escherichia coli outer membrane protease OmpT

Krämer

Brandenburg

Vandeputte-Rutten

et al. 2002

European Journal of Biochemistry

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OmpT is an integral outer membrane protease of Escherichia coli. Overexpression of OmpT in E. coli and subsequent in vitro folding of the produced inclusion bodies yielded protein with a native-like structure. However, enzymatically active protease was only obtained after addition of the outer membrane lipid lipopolysaccharide (LPS). OmpT is the first example of an enzyme that requires LPS for activity. In this study, we investigated the nature of this activation. Circular dichroism analysis showed that binding of LPS did not lead to large structural changes. Titration of OmpT with LPS and determining the resulting OmpT activity with a fluorimetric assay yielded a dissociation constant of 10 )4 M for E. coli K-12 LPS. Determining the dissociation constants for different LPS chemotypes revealed that a fully acylated lipid A part is minimally required for activation of OmpT. The heptose-bound phosphates in the inner core region were also important for activation. The affinity for LPS was not dependent on the concentration of substrate, neither was affinity for the substrate influenced by the concentration of LPS. This indicated that LPS most likely does not act at the level of substrate binding. We hypothesize that LPS induces a subtle conformational change in the protein that is required for obtaining a native active site geometry.

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The effect of the OmpT protease on excision repair in UV-irradiated Escherichia coli

Cited by 8 publications

References 15 publications

Identification of essential acidic residues of outer membrane protease OmpT supports a novel active site

Identification of essential acidic residues of outer membrane protease OmpT supports a novel active site

Identification of active site serine and histidine residues in Escherichia coli outer membrane protease OmpT

Lipopolysaccharide regions involved in the activation of Escherichia coli outer membrane protease OmpT

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