title: Analysis of the link between the redox state and enzymatic activity of the HtrA (DegP) protein from Escherichia coli.
abstract: Bacterial HtrAs are proteases engaged in extracytoplasmic activities during stressful conditions and pathogenesis. A model prokaryotic HtrA (HtrA/DegP from Escherichia coli) requires activation to cleave its substrates efficiently. In the inactive state of the enzyme, one of the regulatory loops, termed LA, forms inhibitory contacts in the area of the active center. Reduction of the disulfide bond located in the middle of LA stimulates HtrA activity in vivo suggesting that this S-S bond may play a regulatory role, although the mechanism of this stimulation is not known. Here, we show that HtrA lacking an S-S bridge cleaved a model peptide substrate more efficiently and exhibited a higher affinity for a protein substrate. An LA loop lacking the disulfide was more exposed to the solvent; hence, at least some of the interactions involving this loop must have been disturbed. The protein without S-S bonds demonstrated lower thermal stability and was more easily converted to a dodecameric active oligomeric form. Thus, the lack of the disulfide within LA affected the stability and the overall structure of the HtrA molecule. In this study, we have also demonstrated that in vitro human thioredoxin 1 is able to reduce HtrA; thus, reduction of HtrA can be performed enzymatically.
authors.affiliation: Department of Biochemistry, Faculty of Biology, University of Gdansk, Gdansk, Poland.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Koper
authors.affiliation: Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, Krakow, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Polit
authors.affiliation: Department of Biochemistry, Faculty of Biology, University of Gdansk, Gdansk, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Sobiecka-Szkatula
authors.affiliation: Department of Molecular and Cellular Biology, Intercollegiate Faculty of Biotechnology of the University of Gdansk and Medical University of Gdansk, Gdansk, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Wegrzyn
authors.affiliation: Department of Life and Environmental Sciences, Universita Politecnica delle Marche, Ancona, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Scire
authors.affiliation: Department of Biochemistry, Faculty of Biology, University of Gdansk, Gdansk, Poland.
authors.forename: Donata
authors.initials: D
authors.lastname: Figaj
authors.affiliation: Department of Molecular and Cellular Biology, Intercollegiate Faculty of Biotechnology of the University of Gdansk and Medical University of Gdansk, Gdansk, Poland.
authors.forename: Leszek
authors.initials: L
authors.lastname: Kadzinski
authors.affiliation: Department of Biochemistry, Faculty of Biology, University of Gdansk, Gdansk, Poland.
authors.forename: Urszula
authors.initials: U
authors.lastname: Zarzecka
authors.affiliation: Department of Biochemistry, Faculty of Biology, University of Gdansk, Gdansk, Poland.
authors.forename: Dorota
authors.initials: D
authors.lastname: Zurawa-Janicka
authors.affiliation: Department of Molecular and Cellular Biology, Intercollegiate Faculty of Biotechnology of the University of Gdansk and Medical University of Gdansk, Gdansk, Poland.
authors.forename: Bogdan
authors.initials: B
authors.lastname: Banecki
authors.affiliation: Department of Biochemistry, Faculty of Chemistry, University of Gdansk, Gdansk, Poland.
authors.forename: Adam
authors.initials: A
authors.lastname: Lesner
authors.affiliation: Department of Life and Environmental Sciences, Universita Politecnica delle Marche, Ancona, Italy.
authors.forename: Fabio
authors.initials: F
authors.lastname: Tanfani
authors.affiliation: Department of Biochemistry, Faculty of Biology, University of Gdansk, Gdansk, Poland.
authors.forename: Barbara
authors.initials: B
authors.lastname: Lipinska
authors.affiliation: Department of Biochemistry, Faculty of Biology, University of Gdansk, Gdansk, Poland.
authors.forename: Joanna
authors.initials: J
authors.lastname: Skorko-Glonek
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004220:Disulfides; D004926:Escherichia coli / Q000201:enzymology*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D033903:Periplasmic Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D013696:Temperature
chemical_list: D004220:Disulfides; D006360:Heat-Shock Proteins; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D033903:Periplasmic Proteins; D011993:Recombinant Fusion Proteins; D006639:Histidine; C061682:DegP protease; D012697:Serine Endopeptidases