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 K(m) value of 36 mM proline and a k(cat)=27 s(-1) were determined for Put1p using an artificial electron acceptor. Put1p also exhibited high activity using ubiquinone-1 (CoQ(1)) as an electron acceptor with a k(cat)=9.6 s(-1) and a K(m) of 33 microM for CoQ(1). 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.
authors.affiliation: Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE 68588, USA.
authors.forename: Srimevan
authors.initials: S
authors.lastname: Wanduragala
authors.forename: Nikhilesh
authors.initials: N
authors.lastname: Sanyal
authors.forename: Xinwen
authors.initials: X
authors.lastname: Liang
authors.forename: Donald F
authors.initials: DF
authors.lastname: Becker
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D011392:Proline / Q000737:chemistry; D011394:Proline Oxidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D014451:Ubiquinone / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014451:Ubiquinone; D011392:Proline; D011394:Proline Oxidase; C025203:Ubiquinone Q1
references.citation: J Biol Chem. 1969 Aug 10;244(15):3999-4006
references.pmid: 4389773
references.citation: Mol Cell Biol. 1986 Jul;6(7):2638-45
references.pmid: 3537723
references.citation: Proc Natl Acad Sci U S A. 1988 Apr;85(7):2036-40
references.pmid: 3353366
references.citation: EMBO J. 2003 Oct 1;22(19):5147-53
references.pmid: 14517252
references.citation: J Bacteriol. 1979 Nov;140(2):504-7
references.pmid: 387738
references.citation: Nature. 1997 Sep 18;389(6648):300-5
references.pmid: 9305847
references.citation: J Nutr. 2008 Oct;138(10):2008S-2015S
references.pmid: 18806116
references.citation: Nat Struct Biol. 2003 Feb;10(2):109-14
references.pmid: 12514740
references.citation: J Proteome Res. 2006 Jul;5(7):1543-54
references.pmid: 16823961
references.citation: Anal Biochem. 1976 Aug;74(2):430-40
references.pmid: 962101
references.citation: J Biol Chem. 2005 Mar 25;280(12):11902-10
references.pmid: 15665328
references.citation: J Bacteriol. 1990 Sep;172(9):4927-35
references.pmid: 1975578
references.citation: Gene. 2002 May 15;290(1-2):1-18
references.pmid: 12062797
references.citation: J Biol Chem. 1993 Apr 25;268(12):8972-9
references.pmid: 8473341
references.citation: Appl Microbiol Biotechnol. 2008 Nov;81(2):211-23
references.pmid: 18802692
references.citation: Biochemistry. 2009 Feb 10;48(5):951-9
references.pmid: 19140736
references.citation: Arch Biochem Biophys. 2002 Dec 1;408(1):131-6
references.pmid: 12485611
references.citation: Amino Acids. 2008 Nov;35(4):719-30
references.pmid: 18369526
references.citation: Infect Immun. 2008 Jul;76(7):3037-44
references.pmid: 18458068
references.citation: Cancer Res. 2009 Aug 15;69(16):6414-22
references.pmid: 19654292
references.citation: J Bacteriol. 1979 Nov;140(2):498-503
references.pmid: 387737
references.citation: Biochemistry. 1979 Jan 23;18(2):331-7
references.pmid: 570409
references.citation: Biochemistry. 1995 Sep 12;34(36):11515-20
references.pmid: 7547881
references.citation: J Biol Chem. 2007 May 11;282(19):14316-27
references.pmid: 17344208
references.citation: J Clin Invest. 1992 Nov;90(5):1679-86
references.pmid: 1430199
references.citation: Mol Cell Biol. 1987 Dec;7(12):4431-40
references.pmid: 3125423
references.citation: EMBO J. 2003 Jul 15;22(14):3503-13
references.pmid: 12853466
references.citation: Oncogene. 2006 Sep 14;25(41):5640-7
references.pmid: 16619034
references.citation: Anal Biochem. 1989 Jul;180(1):136-9
references.pmid: 2817336
references.citation: Clin Chim Acta. 1976 Jan 16;66(2):227-39
references.pmid: 1245071
references.citation: J Biol Chem. 2009 Sep 4;284(36):24115-22
references.pmid: 19574222
references.citation: Biochemistry. 2005 Jun 28;44(25):9130-9
references.pmid: 15966737
references.citation: Cancer Res. 2001 Mar 1;61(5):1810-5
references.pmid: 11280728
references.citation: Annu Rev Biochem. 1980;49:1005-61
references.pmid: 6250440
references.citation: FEBS J. 2007 Nov;274(21):5481-504
references.pmid: 17941859
references.citation: Plant Physiol. 2004 Oct;136(2):3313-8
references.pmid: 15448193
references.citation: Biochemistry. 2004 Oct 5;43(39):12539-48
references.pmid: 15449943
references.citation: Biochemistry. 2001 Apr 17;40(15):4714-21
references.pmid: 11294639
references.citation: Biochemistry. 2008 Aug 26;47(34):8894-901
references.pmid: 18672901