Subunit complementation of thymidylate synthase

Abstract

Each of the two active sites of thymidylate synthase contains amino acid residues contributed by the other subunit. For example, Arg-178 of one monomer binds the phosphate group of the substrate dUMP in the active site of the other monomer [Hardy et al. (1987) Science 235, 448-455]. Inactive mutants of such residues should combine with subunits of other inactive mutants to form heterodimeric hybrids with one functional active site. In vivo and in vitro approaches were used to test this hypothesis. In vivo complementation was accomplished by cotransforming plasmid mixtures encoding pools of inactive Arg-178 mutants and pools of inactive Cys-198 mutants into a host strain deficient in thymidylate synthase. Individual inactive mutants of Arg-178 were also cotransformed with the C198A mutant. Subunit complementation was detected by selection or screening for transformants which grew in the absence of thymidine, and hence produced active enzyme. Many mutants at each position representing a wide variety of size and charge supported subunit complementation. In vitro complementation was accomplished by reversible dissociation and unfolding of mixtures of purified individual inactive Arg-178 and Cys-198 mutant proteins. With the R178F + C198A heterodimer, the Km values for dUMP and CH2H4folate were similar to those of the wild-type enzyme. By titrating C198A with R178F under unfolding-refolding conditions, we were able to calculate the kcat value for the active heterodimer. The catalytic efficiency of the single wild-type active site of the C198A + R178F heterodimer approaches that of the wild-type enzyme.

Intragenic complementation and the structure and function of argininosuccinate lyase

“…The greater activity seen experimentally can be attributed to the 5% of ASL activity exhibited by the D87G homotetramer. This type of complementation, the reconstruction of wild-type active sites, has also been observed in another member of the superfamily, adenylosuccinate lyase [104], as well as in the homotrimeric enzyme aspartate transcarbamoylase [123], and homodimeric proteins glutathione reductase [124], thymidylate synthase [125], mercuric reductase [126], and ribulose bisphosphate carboxylase/oxygenase [127]. The reconstruction of active sites clearly explains the complementation event observed between the Q286R and D87G mutations of ASL.…”

Human argininosuccinate lyase: A structural basis for intragenic complementation

“…The three-dimensional structure of human ASL described herein has enabled us to study the concept of intragenic complementation found in certain ASL-deficient strains and provides experimental proof of Crick and Orgel's initial hypothesis of intragenic complementation (17). Evidence for complementation according to the scheme of statistical regeneration of wild-type active sites also has been observed in vitro for the homodimeric proteins thymidylate synthase (18), ribulose-bisphosphate carboxylase͞oxygenase (19), glutathione reductase (20), and mercuric reductase (21) and the homotrimeric enzyme aspartate transcarbamoylase (22). As intragenic complementation has been implicated in other diseases involving mutations in homomultimeric proteins such as propionic acidemia (23,24) and methylmalonic aciduria (25), a logical extension to heteromultimeric proteins also may apply, making intragenic complementation a much overlooked source of phenotypic and biochemical variation in genetic disease.…”

Construction and characterization of heterodimeric soluble quinoprotein glucose dehydrogenase

“…Several groups achieved separation of heterodimers (hybrid dimers) from both parental homodimers using N-or C-terminal tags of six histidines (His-tag), FLAG, polyarginine (Arg-tail), or polyglutamate (Glu-tail) [16][17][18][19][20][21][22]. We had previously found that Arg-tailed PQQGDH-B showed increased affinity to a cation exchange column and could be effectively separated from wild-type enzyme.…”