Expression, purification, crystallization and preliminary X-ray analysis of ORF60, the small subunit (R2) of ribonucleotide reductase from Kaposi's sarcoma-associated herpesvirus (KSHV).
Ribonucleotide reductase (RNR) is responsible for converting ribonucleotides to deoxyribonucleotides, which are the building blocks of DNA. The enzyme is present in all life forms as well as in some large DNA viruses such as herpesviruses. The alpha-herpesviruses and gamma-herpesviruses encode two class Ia RNR subunits, R1 and R2, while the beta-herpesvirus subfamily only encode an inactive R1 subunit. Here, the crystallization of the R2 subunit of RNR encoded by the ORF60 gene from the oncovirus Kaposi's sarcoma-associated gamma-herpesvirus (KSHV) is reported. These are the first crystals of a viral R2 subunit; the use of in situ proteolysis with chymotrypsin and the addition of hexamine cobalt(III) chloride that were necessary to obtain crystals are described. Optimization of the crystallization conditions yielded crystals that diffracted to 2.0 A resolution. The crystals belonged to space group P2(1), with unit-cell parameters a = 63.9, b = 71.2, c = 71.8 A, alpha = 90, beta = 106.7, gamma = 90 degrees. The data set collected was 95.3% complete, with an R(merge) of 9.6%. There are two molecules in the asymmetric unit, corresponding to a solvent content of 43.4%.
Gurmu
Daniel
D
Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles Väg 2, SE-171 77 Stockholm, Sweden.
Dahlroth
Sue-Li
SL
Haas
Juergen
J
Nordlund
Pär
P
Erlandsen
Heidi
H
D003460:Crystallization; D018360:Crystallography, X-Ray; D015870:Gene Expression; D019288:Herpesvirus 8, Human / Q000201:enzymology*; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D021122:Protein Subunits; D012264:Ribonucleotide Reductases