Journal
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY
Volume 76, Issue 3, Pages 454-461Publisher
TAYLOR & FRANCIS LTD
DOI: 10.1271/bbb.110682
Keywords
Saccharomyces cerevisiae; proline synthesis; gamma-glutamyl kinase; PUA domain; gamma-glutamyl phosphate reductase
Categories
Funding
- Program for Promotion of Basic Research Activities for Innovative Biosciences
- Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented industry
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gamma-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 Delta(1)-pyrroline-5-carboxylate synthetase, which consists of a PUA-free GK domain and a gamma-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.
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