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Metal binding and activity of ribonucleotide reductase protein R2 mutants: Conditions for formation of the mixed manganese-iron cofactor
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2009 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 27, 6532-6539 p.Article in journal (Refereed) Published
Abstract [en]

Class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis (C. tm.) lacks the tyrosyl radical and uses a Mn(IV)-Fe(III) cluster for cysteinyl radical initiation in the large subunit. Here we investigated and compared the metal content and specific activity of the C. tm. wild-type R2 protein and its F127Y mutant, as well as the native mouse R2 protein and its Y177F mutant, all produced as recombinant proteins in Escherichia coli. Our results indicate that the affinity of the RNR R2 proteins for binding metals is determined by the nature of one specific residue in the vicinity of the dimetal site, namely the one that carries the tyrosyl radical in class Ia and Ib R2 proteins. In mouse R2, this tyrosyl residue is crucial for the activity of the enzyme, but in C. tm., the corresponding phenylalanine plays no obvious role in activation or catalysis. However, for the C. tm. wild-type R2 protein to bind Mn and gain high specific activity, there seems to be a strong preference for F over Y at this position. In studies of mouse RNR, we find that the native R2 protein does not bind Mn whereas its Y177F mutant incorporates a significant amount of Mn and exhibits 1.4% of native mouse RNR activity. The observation suggests that a manganese-iron cofactor is associated with the weak activity in this protein.

Place, publisher, year, edition, pages
2009. Vol. 48, no 27, 6532-6539 p.
Keyword [en]
metal, activity, R2 protein, mutant, manganese-iron cofactor
National Category
Biophysics
Research subject
Biophysics; Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-31507DOI: 10.1021/bi900693sISI: 000268137600028PubMedID: 19492792OAI: oai:DiVA.org:su-31507DiVA: diva2:277431
Available from: 2009-11-18 Created: 2009-11-18 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Activation and inhibition of diiron and iron-manganese ribonucleotide reductases
Open this publication in new window or tab >>Activation and inhibition of diiron and iron-manganese ribonucleotide reductases
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Ribonucleotide reductase (RNR) catalyses the reduction of ribonucleotides to deoxyribonucleotides. In conventional class I RNRs the active site is located in the R1 subunit, and the R2 subunit contains a diiron cofactor and a stable tyrosyl radical essential for activity.

Class Ic Chlamydia trachomatis RNR lacks the tyrosyl radical and uses a Mn(IV)Fe(III) cofactor for catalysis. The requirement for metals for RNR activation was studied in C. trachomatis F127Y and Y129F R2, and in mouse wild type and Y177F R2 proteins. The results indicate that R2 affinity for metals is determined by the amino acid located next to the metal site, at the position of the radical carrying tyrosyl. Specifically, R2 proteins that contain phenylalanine in this place bind Mn and Fe, and the tyrosyl containing R2s bind only Fe.

Further results show that C. trachomatis RNR can be inhibited by R2 C-terminal oligopeptides. The highest inhibition was observed for a 20-mer peptide indicating that the oligopeptide inhibition mechanism of class Ic is similar to that of the class Ia and b.

The second part of the thesis deals with class Ia RNR inhibition. The results show that a lanthanum complex containing three 1,10-phenantroline molecules (KP772) which has showed promising cytotoxic activity in cancer cell lines inhibits mouse R2 protein in the presence of external reductants by iron-chelation. It is suggested that KP772 has several synergistic inhibition mechanisms that contribute to its overall anticancer activity. Moreover, other results show that Triapine, a promising chemotherapeutic compound, and its Fe, Ga, Zn, and Cu complexes, inhibit mouse R2 in both reducing and non-reducing conditions. Inhibition by Triapine involves the binding of the drug to the surface of the R2 protein leading to labilization of the Fe-center and subsequent Fe-chelation by Triapine. Formation of the Fe(II)-Triapine complex which reacts with O2 to produce reactive oxygen species results in complete RNR inactivation.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2012. 64 p.
Keyword
class Ic, C. trachomatis, cytotoxicity, KP772, lanthanum, manganese–iron cofactor, metal complex, oligopeptide inhibitor, ribonucleotide reductase, Triapine, tyrosyl radical
National Category
Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-75175 (URN)978-91-7447-499-2 (ISBN)
Public defence
2012-05-31, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
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Available from: 2012-05-09 Created: 2012-04-11 Last updated: 2012-04-16Bibliographically approved

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