Purification and characterization of Put1p from Saccharomyces cerevisiae

Srimevan Wanduragala; Nikhilesh Sanyal; Xinwen Liang; Donald F. Becker

doi:10.1016/j.abb.2010.04.020

Purification and characterization of Put1p from Saccharomyces cerevisiae

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

Research output: Contribution to journal › Article › peer-review

36 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 K_m value of 36mM proline and a k_cat=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 k_cat=9.6s^-1 and a K_m 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 language	English (US)
Pages (from-to)	136-142
Number of pages	7
Journal	Archives of Biochemistry and Biophysics
Volume	498
Issue number	2
DOIs	https://doi.org/10.1016/j.abb.2010.04.020
State	Published - Jun 2010

Keywords

PUT1
Proline dehydrogenase
Proline metabolism
Yeast

ASJC Scopus subject areas

Biophysics
Biochemistry
Molecular Biology

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10.1016/j.abb.2010.04.020

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title = "Purification and characterization of Put1p from Saccharomyces cerevisiae",

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.",

keywords = "PUT1, Proline dehydrogenase, Proline metabolism, Yeast",

author = "Srimevan Wanduragala and Nikhilesh Sanyal and Xinwen Liang and Becker, {Donald F.}",

note = "Funding Information: This research was supported by National Institutes of Health Grant GM061068 and the National Center for Research Resources Grant P20 RR-017675 . This research is a contribution of the University of Nebraska Agricultural Research Division, supported in part by funds provided through the Hatch Act. We thank Dr. Nandakumar Madayiputhiya in the Proteomics/Metabolomics Core Facility for performing mass spectral analysis of Put1p.",

year = "2010",

month = jun,

doi = "10.1016/j.abb.2010.04.020",

language = "English (US)",

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AU - Wanduragala, Srimevan

AU - Sanyal, Nikhilesh

AU - Liang, Xinwen

AU - Becker, Donald F.

N1 - Funding Information: This research was supported by National Institutes of Health Grant GM061068 and the National Center for Research Resources Grant P20 RR-017675 . This research is a contribution of the University of Nebraska Agricultural Research Division, supported in part by funds provided through the Hatch Act. We thank Dr. Nandakumar Madayiputhiya in the Proteomics/Metabolomics Core Facility for performing mass spectral analysis of Put1p.

PY - 2010/6

Y1 - 2010/6

N2 - 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.

AB - 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.

KW - PUT1

KW - Proline dehydrogenase

KW - Proline metabolism

KW - Yeast

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Purification and characterization of Put1p from Saccharomyces cerevisiae

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