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Structural Studies of Microbial Proteins - From Escherichia coli and Herpesviruses
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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.
Keyword [en]
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
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-35674ISBN: 978-91-7155-995-1 (print)OAI: oai:DiVA.org:su-35674DiVA: diva2:287917
Public defence
2010-02-19, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 14:00 (English)
Opponent
Supervisors
Note
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
List of papers
1. Crystal Structure of CaiB, a Type-III CoA Transferase in Carnitine Metabolism
Open this publication in new window or tab >>Crystal Structure of CaiB, a Type-III CoA Transferase in Carnitine Metabolism
2004 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 44, 13996-14003 p.Article in journal (Refereed) Published
Abstract [en]

Carnitine is an important molecule in human metabolism, mainly because of its role in the transport of long-chain fatty acids across the inner mitochondrial membrane. Escherichia coli uses carnitine as a terminal electron acceptor during anaerobic metabolism. Bacteria present in our large intestine break down carnitine that is not absorbed in the small intestine. One part of this catabolic pathway is reversible and can be utilized for bioproduction of large amounts of stereochemically pure l-carnitine, which is used medically for the treatment of a variety of human diseases. Here, we present the crystal structure of the E. coli protein CaiB, which is a member of the recently identified type-III coenzyme A (CoA) transferase family and catalyzes the transfer of the CoA moiety between γ-butyrobetaine−CoA and carnitine forming carnityl-CoA and γ-butyrobetaine. This is the first protein from the carnitine metabolic pathway to be structurally characterized. The structure of CaiB reveals a spectacular fold where two monomers are interlaced to form an interlocked dimer. A molecule of the crystallization buffer bis-(2-hydroxyethyl)imino-tris(hydroxymethyl)methane (bis-tris) is bound in a large pocket located primarily in the small domain, and we propose that this pocket constitutes the binding site for both substrate moieties participating in the CaiB transfer reaction. The binding of CoA to CaiB induces a domain movement that closes the active site of the protein. This is the first observation of a domain movement in the type-III CoA transferase family and can play an important role in coupling substrate binding to initiation of the catalytic reaction.

Identifiers
urn:nbn:se:su:diva-35623 (URN)10.1021/bi048481c (DOI)
Available from: 2010-01-19 Created: 2010-01-19 Last updated: 2017-12-12Bibliographically approved
2. The Crystal Structure of the Bifunctional Deaminase/Reductase RibD of the Riboflavin Biosynthetic Pathway in Escherichia coli: Implications for the Reductive Mechanism
Open this publication in new window or tab >>The Crystal Structure of the Bifunctional Deaminase/Reductase RibD of the Riboflavin Biosynthetic Pathway in Escherichia coli: Implications for the Reductive Mechanism
2007 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 373, no 1, 48-64 p.Article in journal (Refereed) Published
Abstract [en]

We have determined the crystal structure of the bi-functional deaminase/reductase enzyme from Escherichia coli (EcRibD) that catalyzes two consecutive reactions during riboflavin biosynthesis. The polypeptide chain of EcRibD is folded into two domains where the 3D structure of the N-terminal domain (1–145) is similar to cytosine deaminase and the C-terminal domain (146–367) is similar to dihydrofolate reductase. We showed that EcRibD is dimeric and compared our structure to tetrameric RibG, an ortholog from Bacillus subtilis (BsRibG). We have also determined the structure of EcRibD in two binary complexes with the oxidized cofactor (NADP+) and with the substrate analogue ribose-5-phosphate (RP5) and superposed these two in order to mimic the ternary complex. Based on this superposition we propose that the invariant Asp200 initiates the reductive reaction by abstracting a proton from the bound substrate and that the pro-R proton from C4 of the cofactor is transferred to C1 of the substrate. A highly flexible loop is found in the reductase active site (159–173) that appears to control cofactor and substrate binding to the reductase active site and was therefore compared to the corresponding Met20 loop of E. coli dihydrofolate reductase (EcDHFR). Lys152, identified by comparing substrate analogue (RP5) coordination in the reductase active site of EcRibD with the homologous reductase from Methanocaldococcus jannaschii (MjaRED), is invariant among bacterial RibD enzymes and could contribute to the various pathways taken during riboflavin biosynthesis in bacteria and yeast.

Identifiers
urn:nbn:se:su:diva-35628 (URN)10.1016/j.jmb.2006.12.009 (DOI)
Available from: 2010-01-19 Created: 2010-01-19 Last updated: 2017-12-12Bibliographically approved
3. The crystal structure of the protein YhaK from Escherichia coli reveals a new subclass of redox sensitive enterobacterial bicupins
Open this publication in new window or tab >>The crystal structure of the protein YhaK from Escherichia coli reveals a new subclass of redox sensitive enterobacterial bicupins
Show others...
2009 (English)In: Proteins: Structure, Function, and Genetics, ISSN 0887-3585, E-ISSN 1097-0134, 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.

Identifiers
urn:nbn:se:su:diva-17352 (URN)10.1002/prot.22128 (DOI)000261757900003 ()18561187 (PubMedID)
Available from: 2009-01-14 Created: 2009-01-14 Last updated: 2017-12-13Bibliographically approved
4. Crystal structure of the shutoff and exonuclease protein from the oncogenic Kaposi's sarcoma-associated herpesvirus
Open this publication in new window or tab >>Crystal structure of the shutoff and exonuclease protein from the oncogenic Kaposi's sarcoma-associated herpesvirus
Show others...
2009 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 276, no 22, 6636-6645 p.Article in journal (Refereed) Published
Abstract [en]

The Kaposi's sarcoma-associated herpesvirus protein SOX (shut off and exonuclease) and its Epstein–Barr virus homolog, BGLF5, are active during the early lytic phase and belong to the alkaline nuclease family. Both proteins have been shown to be bifunctional, being responsible for DNA maturation as well as host shutoff at the mRNA level. We present the crystal structure of SOX determined at 1.85 Å resolution. By modeling DNA binding, we have identified catalytic residues that explain the preferred 5'-exonuclease activity of the alkaline nucleases. The presence of a crevice suitable for binding duplex DNA supports a role for herpes alkaline nucleases in recombination events preceding packaging of viral DNA. Direct interaction with dsDNA is supported by oligonucleotide binding data. Mutations specifically affecting host shutoff map to a surface region of the N-terminal domain, implying an essential role in protein–protein interactions, and link the RNase activity of the enzyme to mRNA degradation pathways.

Identifiers
urn:nbn:se:su:diva-25561 (URN)10.1111/j.1742-4658.2009.07374.x (DOI)000271057200021 ()
Note
Part of urn:nbn:se:su:diva-8288Available from: 2008-10-30 Created: 2008-10-23 Last updated: 2017-12-13Bibliographically approved
5. Expression,
 purification,
 crystallization
 and
 preliminary
 X­ray
 analysis
 of
 ORF60,
 the
 small
 subunit
 (R2)
 of
 ribonucleotide
 reductase
 from
 Kaposi’s
 sarcoma­associated
herpesvirus
(KSHV)
Open this publication in new window or tab >>Expression,
 purification,
 crystallization
 and
 preliminary
 X­ray
 analysis
 of
 ORF60,
 the
 small
 subunit
 (R2)
 of
 ribonucleotide
 reductase
 from
 Kaposi’s
 sarcoma­associated
herpesvirus
(KSHV)
Show others...
(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:su:diva-35646 (URN)
Available from: 2010-01-19 Created: 2010-01-19 Last updated: 2010-01-20Bibliographically approved

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