Physiological characterization of the ARO10-dependent, broad-substrate-specificity 2-oxo acid decarboxylase activity of Saccharomyces cerevisiae.
Aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae CEN.PK113-7D were grown with different nitrogen sources. Cultures grown with phenylalanine, leucine, or methionine as a nitrogen source contained high levels of the corresponding fusel alcohols and organic acids, indicating activity of the Ehrlich pathway. Also, fusel alcohols derived from the other two amino acids were detected in the supernatant, suggesting the involvement of a common enzyme activity. Transcript level analysis revealed that among the five thiamine-pyrophospate-dependent decarboxylases (PDC1, PDC5, PDC6, ARO10, and THI3), only ARO10 was transcriptionally up-regulated when phenylalanine, leucine, or methionine was used as a nitrogen source compared to growth on ammonia, proline, and asparagine. Moreover, 2-oxo acid decarboxylase activity measured in cell extract from CEN.PK113-7D grown with phenylalanine, methionine, or leucine displayed similar broad-substrate 2-oxo acid decarboxylase activity. Constitutive expression of ARO10 in ethanol-limited chemostat cultures in a strain lacking the five thiamine-pyrophosphate-dependent decarboxylases, grown with ammonia as a nitrogen source, led to a measurable decarboxylase activity with phenylalanine-, leucine-, and methionine-derived 2-oxo acids. Moreover, even with ammonia as the nitrogen source, these cultures produced significant amounts of the corresponding fusel alcohols. Nonetheless, the constitutive expression of ARO10 in an isogenic wild-type strain grown in a glucose-limited chemostat with ammonia did not lead to any 2-oxo acid decarboxylase activity. Furthermore, even when ARO10 was constitutively expressed, growth with phenylalanine as the nitrogen source led to increased decarboxylase activities in cell extracts. The results reported here indicate the involvement of posttranscriptional regulation and/or a second protein in the ARO10-dependent, broad-substrate-specificity decarboxylase activity.
Vuralhan
Zeynep
Z
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
Luttik
Marijke A H
MA
Tai
Siew Leng
SL
Boer
Viktor M
VM
Morais
Marcos A
MA
Schipper
Dick
D
Almering
Marinka J H
MJ
Kötter
Peter
P
Dickinson
J Richard
JR
Daran
Jean-Marc
JM
Pronk
Jack T
JT
D002262:Carboxy-Lyases / Q000378:metabolism*; D003470:Culture Media; D003653:Decarboxylation; D015966:Gene Expression Regulation, Fungal; D005947:Glucose / Q000378:metabolism; D007930:Leucine / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism*; D010649:Phenylalanine / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000254:growth & development* / Q000378:metabolism / Q000502:physiology; D013379:Substrate Specificity; D015854:Up-Regulation
D003470:Culture Media; D010649:Phenylalanine; D008715:Methionine; D002262:Carboxy-Lyases; C109058:phenylpyruvate decarboxylase; D007930:Leucine; D005947:Glucose