RRC ID 2184
Author Terao Y, Nakamori S, Takagi H.
Title Gene dosage effect of L-proline biosynthetic enzymes on L-proline accumulation and freeze tolerance in Saccharomyces cerevisiae.
Journal Appl. Environ. Microbiol.
Abstract We have previously reported that L-proline has cryoprotective activity in Saccharomyces cerevisiae. A freeze-tolerant mutant with L-proline accumulation was recently shown to carry an allele of the PRO1 gene encoding gamma-glutamyl kinase, which resulted in a single amino acid substitution (Asp154Asn). Interestingly, this mutation enhanced the activities of gamma-glutamyl kinase and gamma-glutamyl phosphate reductase, both of which catalyze the first two steps of L-proline synthesis and which together may form a complex in vivo. Here, we found that the Asp154Asn mutant gamma-glutamyl kinase was more thermostable than the wild-type enzyme, which suggests that this mutation elevated the apparent activities of two enzymes through a stabilization of the complex. We next examined the gene dosage effect of three L-proline biosynthetic enzymes, including Delta(1)-pyrroline-5-carboxylate reductase, which converts Delta(1)-pyrroline-5-carboxylate into L-proline, on L-proline accumulation and freeze tolerance in a non-L-proline-utilizing strain. Overexpression of the wild-type enzymes has no influence on L-proline accumulation, which suggests that the complex is very unstable in nature. However, co-overexpression of the mutant gamma-glutamyl kinase and the wild-type gamma-glutamyl phosphate reductase was effective for L-proline accumulation, probably due to a stabilization of the complex. These results indicate that both enzymes, not Delta(1)-pyrroline-5-carboxylate reductase, are rate-limiting enzymes in yeast cells. A high tolerance for freezing clearly correlated with higher levels of L-proline in yeast cells. Our findings also suggest that, in addition to its cryoprotective activity, intracellular L-proline could protect yeast cells from damage by oxidative stress. The approach described here provides a valuable method for breeding novel yeast strains that are tolerant of both freezing and oxidative stresses.
Volume 69(11)
Pages 6527-32
Published 2003-11
PMID 14602584
PMC PMC262311
MeSH Freezing Gene Dosage* Gene Expression Regulation, Fungal* Heat-Shock Response Molecular Sequence Data Oxidative Stress Phosphotransferases (Carboxyl Group Acceptor) / genetics Proline / biosynthesis* Proline / metabolism Saccharomyces cerevisiae / enzymology* Saccharomyces cerevisiae / genetics Saccharomyces cerevisiae / growth & development Saccharomyces cerevisiae / physiology* Saccharomyces cerevisiae Proteins / genetics Saccharomyces cerevisiae Proteins / metabolism Sequence Analysis, DNA
IF 4.077
Times Cited 47
WOS Category BIOTECHNOLOGY & APPLIED MICROBIOLOGY MICROBIOLOGY
Resource
Yeast BYP1804(pCgHIS3)