Ohr (Organic Hydroperoxide Resistance Protein) Possesses a Previously Undescribed Activity, Lipoyl-dependent Peroxidase

José R.R Cussiol,Thiago G.P Alegria,Luke I Szweda,Luis E.S Netto

doi:10.1074/jbc.m110.117283

José R.R Cussiol, Thiago G.P Alegria + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m110.117283

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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.

Highlights

Ohr belongs to a family of proteins that comprise osmotically inducible protein (OsmC)
It was demonstrated that OsmC enzymes possess thiol-dependent peroxidase activity and share the same structural fold with Ohr proteins [15, 16]
We show that peroxidase activity of Ohr from X. fastidiosa is supported by lipoylated proteins but not by thioredoxin and glutathione systems

Summary

Introduction

Ohr belongs to a family of proteins that comprise osmotically inducible protein (OsmC). We show that peroxidase activity of Ohr from X. fastidiosa is supported by lipoylated proteins but not by thioredoxin and glutathione systems. For the expression of lipoylated enzymes from the strains containing pET15b/lpdA, pET15b/PDHB, and pET15b/sucB, 300 ␮g/ml lipoic acid was added to the culture medium to ensure maximum lipoylation of these proteins.

Results

Conclusion