Purification and characterization of Put1p from Saccharomyces cerevisiae

Srimevan Wanduragala, Nikhilesh Sanyal, Xinwen Liang, Donald F Becker

Research output: Contribution to journalArticle

29 Scopus citations

Abstract

In Saccharomyces cerevisiae, the PUT1 and PUT2 genes are required for the conversion of proline to glutamate. The PUT1 gene encodes Put1p, a proline dehydrogenase (PRODH) enzyme localized in the mitochondrion. Put1p was expressed and purified from Escherichia coli and shown to have a UV-visible absorption spectrum that is typical of a bound flavin cofactor. A Km value of 36mM proline and a kcat=27s-1 were determined for Put1p using an artificial electron acceptor. Put1p also exhibited high activity using ubiquinone-1 (CoQ1) as an electron acceptor with a kcat=9.6s-1 and a Km of 33μM for CoQ1. In addition, knockout strains of the electron transfer flavoprotein (ETF) homolog in S. cerevisiae were able to grow on proline as the sole nitrogen source demonstrating that ETF is not required for proline utilization in yeast.

Original languageEnglish (US)
Pages (from-to)136-142
Number of pages7
JournalArchives of Biochemistry and Biophysics
Volume498
Issue number2
DOIs
StatePublished - Jun 1 2010

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Keywords

  • PUT1
  • Proline dehydrogenase
  • Proline metabolism
  • Yeast

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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