CATALYTICALLY ACTIVE CROSS-SPECIES HETERODIMERS OF THYMIDYLATE SYNTHASE

Thymidylate synthase (TS) is a highly conserved homodimeric enzyme wit h two active sites, each of which contains amino acid residues from bo th subunits. We show that the conservation at the subunit interface be tween Escherichia coli TS and Lactobacillus casei TS is sufficient to permit the formation of a cross-species heterodimer between subunits o f E. coli TS and L. casei TS. Heterodimer formation was monitored by t he generation of catalytic activity when combinations of inactive E. c oli homodimers and inactive L. casei homodimers were mixed under condi tions 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)K4 8Q, (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 t he mutant enzymes tested were able to form catalytically active cross- species heterodimers. (Lc)C198A and (Ec)R126Q were cotransformed on co mpatible plasmids into a thymine-requiring E. coli host, and this comb ination was able to form sufficient active TS in vivo to support growt h. Titration of (Ec)R126Q with (Lc)C198A showed that the cross-species heterodimer formed with the same probability as the intraspecies homo dimers in the refolding mixture. The single active site formed by this pair has k(cat) and K(m) values similar to those of an intraspecies h eterodimer.