Characterization and mutational studies of equine infectious anemia virus dUTPase.
The macrophage tropic lentivirus, equine infectious anemia virus (EIAV), encodes a dUTPase in the pol gene that is required for efficient replication in macrophages. Two naturally occurring variants of the enzyme were expressed as recombinant proteins in Escherichia coli; metal chelate affinity chromatography was used to purify histidine-tagged recombinant enzymes to greater than 80% homogeneity in a single chromatographic step. Biochemical and enzymatic analyses of these preparations suggest that this method yields dUTPase that is suitable for detailed mutational analysis. Specific activities of preparations ranged from 4 x 10(3) to 5 x 10(4) units/mg. Recombinant EIAV dUTPase was highly specific for dUTP with a Km in the range of 3 to 8 microM. The enzyme was sensitive to inhibition by dUDP with little inhibition by other nucleotides or the reaction products, dUMP and PPi. The subunit organization of recombinant EIAV dUTPase was probed by gel filtration, glycerol gradient centrifugation, and chemical cross-linking, and is a trimer. We have begun mutational analyses by targeting a conserved domain present at the carboxyl terminus of all dUTPases that shares high homology to the phosphate binding loops (P-loops) of a number of ATP- and GTP-binding phosphatases. The P-loop-like motif of dUTPases is glycine rich but lacks the invariant lysine found in authentic P-loops. Deletion of this motif leads to loss of dUTPase activity; a series of point mutations that have been shown to inactivate authentic P-loops also abolish EIAV dUTPase activity.
Shao
H
H
Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, OH 44106-4960, USA.
Robek
M D
MD
Threadgill
D S
DS
Mankowski
L S
LS
Cameron
C E
CE
Fuller
F J
FJ
Payne
S L
SL
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology; D006639:Histidine / Q000737:chemistry; D004860:Infectious Anemia Virus, Equine / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011755:Pyrophosphatases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D013379:Substrate Specificity
D011994:Recombinant Proteins; D006639:Histidine; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase