We overexpressed and purified from Escherichia coli the dihydrofolate reductase (DHFR) of the gammaherpesviruses human herpesvirus 8 (HHV-8), herpesvirus saimiri (HVS), and rhesus rhadmovirus (RRV). All three enzymes proved catalytically active. The K-m value of HHV-8 DHFR for dihydrofolate (DHF) was 2.02 +/- 0.44 muM, that of HVS DHFR was 4.31 +/- 0.56 muM, and that of RRV DHFR is 7.09 +/- 0.11 muM. These values are approximately 5-15-fold higher than the K-m value reported for the human DHFR. The K-m value of HHV-8 DHFR for NADPH was 1.31 +/- 0.23 muM, that of HVS DHFR was 3.78 +/- 0.61 muM, and that of RRV DHFR was 7.47 +/- 0.59 muM. These values are similar or slightly higher than the corresponding K-m value of the human enzyme. Methotrexate, aminopterin, trimethoprim, pyrimethamine, and N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523), all well-known folate antagonists, inhibited the DHFR activity of the three gamma herpesviruses competitively with respect to DHF but proved markedly less inhibitory to the viral than towards the human enzyme. (C) 2002 Elsevier Science (USA). All rights reserved.