Ohr (Organic hydroperoxide resistance protein) Possesses a Previously Undescribed Activity: Lipoyldependent Peroxidase

Abstract

The Ohr (organic hydroperoxide resistance) family of 15-kDa Cys-based, thiol-dependent peroxidases is central to the bacterial response to stress induced by organic hydroperoxides but not by hydrogen peroxide. Ohr has a unique three-dimensional structure and requires dithiols, but not monothiols, to support its activity. However, the physiological reducing system of Ohr has not yet been identified. Here we show that lipoylated enzymes present in the bacterial extracts of Xylella fastidiosa interacted physically and functionally with this Cys-based peroxidase, whereas thioredoxin and glutathione systems failed to support Ohr peroxidase activity. Furthermore, we could reconstitute in vitro three lipoyl-dependent systems as the Ohr physiological reducing systems. We also showed that OsmC from Escherichia coli, an orthologue of Ohr from Xylella fastidiosa, is specifically reduced by lipoyl-dependent systems. These results represent the first description of a Cys-based peroxidase that is directly reduced by lipoylated enzymes.

Thiol‐based switch mechanism of virulence regulator AphB modulates oxidative stress response in Vibrio cholerae

“…Sequence analysis revealed that the plasmid harbored a gene annotated as ohrA (vca1006), which is predicted to encode a putative organic hydroperoxide resistance protein. In other bacteria, OhrA has been shown to be a lipoyl-dependent peroxidase, reducing both inorganic and organic hydroperoxides to less toxic metabolites at its cysteine thiol (Lesniak et al, 2002;Cussiol et al, 2010). OhrA of V. cholerae is highly homologous to these well-studied OhrA proteins, and in particular contains two conserved cysteine residues (Supporting Information Fig.…”

Mecanismos de captação de heme em Chromobacterium violaceum

“…Inicialmente, os pré-inóculos das linhagens foram diluídos para DO600 0,05 em 500 mL de LB suplementado com ampicilina. As culturas foram então cultivadas a 37 ºC sob agitação de 250 rpm até ser atingida DO600 0,4 -0,6.Posteriormente, IPTG (IPTG -Isopropyl b-D-1-thiogalactopyranoside) foi adicionado aos cultivos (1 mM para His-ChuP; 0,5 mM para His-ChuS) e estes induzidos (37 ºC sob 250 rpm por 2 horas para His-ChuP; 20 ºC sob 60 rpm por 24 horas para His-ChuP)(CUSSIOL et al, 2010). Após indução, as culturas foram centrifugadas a 14000x g por 5 minutos a 4 ºC e os pellets obtidos foram ressuspendidos em água Milli-Q, centrifugados e armazenados a -20 ºC.…”