Catalytically active cross-species heterodimers of thymidylate synthase.
Thymidylate synthase (TS) is a highly conserved homodimeric enzyme with two active sites, each of which contains amino acid residues from both subunits. We show that the conservation at the subunit interface between Escherichia coli TS and Lactobacillus casei TS is sufficient to permit the formation of a cross-species heterodimer between subunits of E. coli TS and L. casei TS. Heterodimer formation was monitored by the generation of catalytic activity when combinations of inactive E. coli homodimers and inactive L. casei homodimers were mixed under conditions of reversible unfolding and dissociation. The inactive L. casei mutant enzymes (Lc)C198A, (Lc)C198L, and (Lc)V316Am were tested as Arg donors to the active sites of the inactive E. coli mutant enzymes (Ec)R126Q and (Ec)R126E, while the inactive E. coli mutant enzymes (Ec)K48Q, (Ec)C146S, (Ec)R166Q, and (Ec)I264Am were tested as Arg donors to the active site of inactive (Lc)R178F. Except for (Lc)V316Am, all of the mutant enzymes tested were able to form catalytically active cross-species heterodimers. (Lc)C198A and (Ec)R126Q were cotransformed on compatible plasmids into a thymine-requiring E. coli host, and this combination was able to form sufficient active TS in vivo to support growth. Titration of (Ec)R126Q with (Lc)C198A showed that the cross-species heterodimer formed with the same probability as the intraspecies homodimers in the refolding mixture. The single active site formed by this pair has kcat and Km values similar to those of an intraspecies heterodimer.
Greene
P J
PJ
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
Maley
F
F
Pedersen-Lane
J
J
Santi
D V
DV
D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology; D005468:Fluorodeoxyuridylate / Q000378:metabolism; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology; D046911:Macromolecular Substances; D016296:Mutagenesis; D017510:Protein Folding; D013940:Thymidylate Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D046911:Macromolecular Substances; D005468:Fluorodeoxyuridylate; D013940:Thymidylate Synthase