title: Functional analysis of the C-terminal region of γ-glutamyl kinase of Saccharomyces cerevisiae.
abstract: γ-Glutamyl kinase (GK) is the rate-limiting enzyme in proline synthesis in microorganisms. Most microbial GKs contain an N-terminal kinase domain and a C-terminal pseudouridine synthase and archaeosine transglycosylase (PUA) domain. In contrast, higher eukaryotes possess a bifunctional Δ(1)-pyrroline-5-carboxylate synthetase, which consists of a PUA-free GK domain and a γ-glutamyl phosphate reductase (GPR) domain. Here, to examine the role of the C-terminal region, including the PUA domain of Saccharomyces cerevisiae GK, we constructed a variety of truncated yeast GK and GK/GPR fusion proteins from which the C-terminal region was deleted. A complementation test in Escherichia coli and S. cerevisiae and enzymatic analysis of recombinant proteins revealed that a 67-residue linker sequence between a 255-residue kinase domain and a 106-residue PUA domain is essential for GK activity. It also appeared that 67 or more residues of the C-terminal region, not the PUA domain itself, are required for the full display of GK activity. Further, the GK/GPR fusion protein was functional in E. coli, but decreased stability and Mg-binding ability as compared to wild-type GK. These results suggest that the C-terminal region of S. cerevisiae GK is involved in the folding and/or the stability of the kinase domain.
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Kaino
authors.forename: Yumi
authors.initials: Y
authors.lastname: Tasaka
authors.forename: Yuki
authors.initials: Y
authors.lastname: Tatehashi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Takagi
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D017384:Sequence Deletion
chemical_list: D011993:Recombinant Fusion Proteins; D017851:Phosphotransferases (Carboxyl Group Acceptor); C020650:glutamate 5-kinase