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The crystal structure of the protein YhaK from Escherichia coli reveals a new subclass of redox sensitive enterobacterial bicupins
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2009 (English)In: Proteins: Structure, Function, and Genetics, ISSN 0887-3585, Vol. 74, no 1, 18-31 p.Article in journal (Refereed) Published
Abstract [en]

YhaK is a protein of unknown function found in low abundance in the cytosol of Escherichia coli. DNA array studies have revealed that YhaK is strongly up-regulated by nitroso-glutathione (GSNO) and also displays a 12-fold increase in expression during biofilm growth of E. coli 83972 and VR50 in human urine. We have determined the YhaK crystal structure and demonstrated that in vitro YhaK is a good marker for monitoring oxidative stresses in E. coli. The YhaK protein structure shows a bicupin fold where the two cupin domains are crosslinked with one intramolecular disulfide bond (Cys10 to Cys204). We found that the third cysteine in YhaK, Cys122, is oxidized to a sulfenic acid. Two chloride ions are found in the structure, one close to the reactive Cys122, and the other on a hydrophobic surface close to a symmetry-related molecule. There are major structural differences at the N-terminus of YhaK compared with similar structures that also display the bicupin fold (YhhW and hPirin). YhaK showed no quercetinase and peroxidase activity. However, reduced YhaK was very sensitive to reactive oxygen species (ROS). The complete, functional E. coli glutaredoxin or thioredoxin systems protected YhaK from oxidation. E. coli thioredoxin reductase and NADPH produced ROS and caused oxidation and oligomerization of reduced YhaK. Taken together, we propose that YhaK is the first of a new sub-class of bicupins that lack the canonical cupin metal-binding residues of pirins and may be involved in chloride binding and/or sensing of oxidative stress in enterobacteria.

Place, publisher, year, edition, pages
2009. Vol. 74, no 1, 18-31 p.
URN: urn:nbn:se:su:diva-17352DOI: 10.1002/prot.22128ISI: 000261757900003PubMedID: 18561187OAI: diva2:183873
Available from: 2009-01-14 Created: 2009-01-14 Last updated: 2010-01-20Bibliographically approved
In thesis
1. Structural Studies of Microbial Proteins - From Escherichia coli and Herpesviruses
Open this publication in new window or tab >>Structural Studies of Microbial Proteins - From Escherichia coli and Herpesviruses
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Structure biology concerns the study of the molecular structures of biological macromolecules, such as proteins, and how these relate to the function. Protein structures are also of importance in structure-based drug design. In this thesis, the work has been carried out in two different projects. The first project concerns structural studies of proteins from the bacterium Escherichia coli and the second of proteins from five different herpesviruses.

 The E. coli project resulted in the structural characterization of three proteins: CaiB, RibD, and YhaK. CaiB is a type-III CoA transferase involved in the metabolism of carnitine. Its molecular structure revealed a spectacular fold where two monomers were interlaced forming an interlocked dimer. RibD, a bi-functional enzyme, catalyzes two consecutive reactions during riboflavin biosynthesis. In an attempt to characterize the mechanism of action of the N-terminal reductase domain, the structure of RibD was also determined in two binary complexes with the oxidized cofactor, NADP+, and with the substrate analogue ribose-5-phosphate. YhaK is a protein of unknown function normally found in low abundance in the cytosol of E. coli and was previously annotated to be a member of the Pirin family. However, some structural features seem to distinguish YhaK from these other Pirin proteins and we showed that YhaK might be regulated by reactive oxygen species.

 The Herpesvirus project resulted in the structural determination of two proteins, the SOX protein and ORF60 from Kaposi’s sarcoma associated herpesvirus (KSHV). SOX, a bi-functional shutoff and exonuclease protein, is involved in the maturation and packaging of the viral genome into the viral capsid and in the host shutoff of cellular proteins at the mRNA level. The SOX structure was also used for modeling DNA binding. The crystallization and preliminary structural studies of ORF60, the small R2 subunit of the ribonucleotide reductase (RNR) from KSHV is also discussed.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2010. 64 p.
CaiB, RibD, YhaK, SOX, ORF60, Herpesvirus, E. coli
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
urn:nbn:se:su:diva-35674 (URN)978-91-7155-995-1 (ISBN)
Public defence
2010-02-19, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 14:00 (English)
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 5: Manuscript.Available from: 2010-01-28 Created: 2010-01-19 Last updated: 2010-01-27Bibliographically approved

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