A Kinetic Study on the Influence of Nucleoside Triphosphate Effectors on Subunit Interaction in Mouse Ribonucleotide Reductase†

Herpesvirus ribonucleotide reductases, essential for the de novo synthesis of viral DNA, are composed of two non-identical subunits, termed R1 and R2. The U28 ORF from human herpesvirus-7 has been classified, by sequence comparisons, as a homologue of the R1 subunit from ribonucleotide reductase but no R2 ORF is present. Detailed analysis of the U28 amino acid sequence indicated that a number of essential R1 catalytic residues are absent. Cloning and expression of the U28 protein in E. coli and its subsequent characterization in subunit interaction and enzyme activity assays confirmed that it is not a functional equivalent of a herpesvirus R1. In the absence of the R2 gene, we propose that the R1 ORF has evolved a distinct, as yet unidentified, function not only in human herpesvirus-7 but also in other human betaherpesviruses.

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Characterization of heterosubunit complexes formed by the R1 and R2 subunits of herpes simplex virus 1 and equine herpes virus 4 ribonucleotide reductase

Biochemical Journal ◽

10.1042/bj3470097 ◽

2000 ◽

Vol 347 (1) ◽

pp. 97-104

Author(s):

Yunming SUN ◽

Joe CONNER

Keyword(s):

Enzyme Activity ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Ribonucleotide Reductase ◽

Herpes Virus ◽

Subunit Interaction ◽

Herpes Simplex Virus 1 ◽

Herpès Virus ◽

Simplex Virus ◽

Hsv 1

We report on the separate PCR cloning and subsequent expression and purification of the large (R1) and small (R2) subunits from equine herpes virus type 4 (EHV-4) ribonucleotide reductase. The EHV-4 R1 and R2 subunits reconstituted an active enzyme and their abilities to complement the R1 and R2 subunits from the closely related herpes simplex virus 1 (HSV-1) ribonucleotide reductase, with the use of subunit interaction and enzyme activity assays, were analysed. Both EHV-4 R1/HSV-1 R2 and HSV-1 R1/EHV-4 R2 were able to assemble heterosubunit complexes but, surprisingly, neither of these complexes was fully active in enzyme activity assays; the EHV-4 R1/HSV-1 R2 and HSV-1 R1/EHV-4 R2 enzymes had 50% and 5% of their respective wild-type activities. Site-directed mutagenesis was used to alter two non-conserved residues located within the highly conserved and functionally important C-termini of the EHV-4 and HSV-1 R1 proteins. Mutation of Pro-737 to Lys and Lys-1084 to Pro in EHV-4 and HSV-1 R1 respectively had no effects on subunit assembly. Mutation of Pro-737 to Lys in EHV-4 R1 decreased enzyme activity by 50%; replacement of Lys-1084 by Pro in HSV-1 R1 had no effect on enzyme activity. Both alterations failed to restore full enzyme activities to the heterosubunit enzymes. Therefore probably neither of these amino acids has a direct role in catalysis. However, mutation of the highly conserved Tyr-1111 to Phe in HSV-1 R1 inactivated enzyme activity without affecting subunit interaction.

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1H NMR Studies of Mouse Ribonucleotide Reductase: The R2 Protein Carboxyl-Terminal Tail, Essential for Subunit Interaction, Is Highly Flexible but becomes Rigid in the Presence of Protein R1

Biochemistry ◽

10.1021/bi00176a013 ◽

1994 ◽

Vol 33 (10) ◽

pp. 2838-2842 ◽

Cited By ~ 33

Author(s):

Per Olof Lycksell ◽

Rolf Ingemarson ◽

Ralph Davis ◽

Astrid Graeslund ◽

Lars Thelander

Keyword(s):

1H Nmr ◽

Ribonucleotide Reductase ◽

Subunit Interaction ◽

Nmr Studies ◽

Carboxyl Terminal

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A Novel One-Pot Enzyme Cascade for the Biosynthesis of Cladribine Triphosphate

Biomolecules ◽

10.3390/biom11030346 ◽

2021 ◽

Vol 11 (3) ◽

pp. 346

Author(s):

Julia Frisch ◽

Tin Maršić ◽

Christoph Loderer

Keyword(s):

Ribonucleotide Reductase ◽

Nucleoside Triphosphate ◽

Adenine Phosphoribosyltransferase ◽

Phosphoribosyl Pyrophosphate ◽

One Pot ◽

Nucleotide Biosynthesis ◽

Enzyme Cascade ◽

Anti Cancer ◽

Enzyme Cascades ◽

Ribose Moiety

Cladribine triphosphate is the active compound of the anti-cancer and multiple sclerosis drug Mavenclad (cladribine). Biosynthesis of such non-natural deoxyribonucleotides is challenging but important in order to study the pharmaceutical modes of action. In this study, we developed a novel one-pot enzyme cascade for the biosynthesis of cladribine triphosphate, starting with the nucleobase 2Cl-adenine and the generic co-substrate phosphoribosyl pyrophosphate. The cascade is comprised of the three enzymes, namely, adenine phosphoribosyltransferase (APT), polyphosphate kinase (PPK), and ribonucleotide reductase (RNR). APT catalyzes the binding of the nucleobase to the ribose moiety, followed by two consecutive phosphorylation reactions by PPK. The formed nucleoside triphosphate is reduced to the final product 2Cl-deoxyadenonsine triphosphate (cladribine triphosphate) by the RNR. The cascade is feasible, showing comparative product concentrations and yields to existing enzyme cascades for nucleotide biosynthesis. While this study is limited to the biosynthesis of cladribine triphosphate, the design of the cascade offers the potential to extend its application to other important deoxyribonucleotides.

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