Regulation of D-amino acid oxidase expression in the yeast Rhodotorula gracilis.
Rhodotorula gracilis is a oleaginous yeast which utilizes D-amino acids as a source of carbon and/or nitrogen. D-amino acid oxidase (DAAO), which converts D-amino acids in the corresponding alpha-keto acids and ammonia, is the first enzyme involved in the catabolism of D-amino acids. DAAO activity is induced by the presence of D-alanine, but the presence of the L-isomer prevents induction by inhibiting the transport of D-alanine into cells. To understand how DAAO expression is regulated, R. gracilis cells were grown on media containing different nitrogen and/or carbon sources. As a general rule, the level of DAAO mRNA reached a maximum after 15 h growth and preceded by approximately 6 h the maximum level of DAAO activity. The inducer D-alanine acts by increasing the rate of DAAO mRNA transcription: the increase in DAAO expression is due essentially to de novo synthesis. The presence of a supplemental carbon source (e.g. succinate or glucose) does not repress DAAO expression. Ammonium sulphate appears to have a negative effect on DAAO mRNA translation and on the expression of DAAO activity: DAAO is only partially active when the yeast is grown in the presence of D-alanine and ammonium sulphate. The best expression of DAAO activity was obtained by growing the cells for 12 h at 30 degrees C in the presence of glucose and D-alanine using cells pre-cultured for 10 h on glucose and L-alanine (0.99 U/mg protein, corresponding to approximately 1.0% total proteins in the crude extract). Under these growth conditions a six-fold increase in DAAO production was achieved.
Molla
Gianluca
G
Department of Structural and Functional Biology, University of Insubria, via J.H. Dunant 3, 21100 Varese, Italy.
Motteran
Laura
L
Piubelli
Luciano
L
Pilone
Mirella S
MS
Pollegioni
Loredano
L
D000409:Alanine / Q000378:metabolism; D000645:Ammonium Sulfate / Q000378:metabolism; D015152:Blotting, Northern; D015153:Blotting, Western; D003605:D-Amino-Acid Oxidase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D015966:Gene Expression Regulation, Fungal / Q000502:physiology*; D005947:Glucose / Q000378:metabolism; D012331:RNA, Fungal / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012248:Rhodotorula / Q000201:enzymology / Q000235:genetics / Q000502:physiology*
D005656:Fungal Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger; D003605:D-Amino-Acid Oxidase; D005947:Glucose; D000409:Alanine; D000645:Ammonium Sulfate