TY - JOUR
T1 - Purification and characterization of Put1p from Saccharomyces cerevisiae
AU - Wanduragala, Srimevan
AU - Sanyal, Nikhilesh
AU - Liang, Xinwen
AU - Becker, Donald F.
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.
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U2 - 10.1016/j.abb.2010.04.020
DO - 10.1016/j.abb.2010.04.020
M3 - Article
C2 - 20450881
AN - SCOPUS:77953541273
SN - 0003-9861
VL - 498
SP - 136
EP - 142
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 2
ER -