Investigation of substrate binding and product stereochemistry issues in two linoleate 9-lipoxygenases.
Herein we characterize the Arabidopsis thaliana AtLOX1 and tomato (Solanum lycopersicum) LOXA proteins as linoleate 9S-lipoxygenases (9-LOX), and use the enzymes to test a model that predicts a relationship between substrate binding orientation and product stereochemistry. The cDNAs were heterologously expressed in E. coli and the proteins partially purified by nickel affinity chromatography using a N-terminal (His)(6)-tag. Both enzymes oxygenated linoleic acid almost exclusively to the 9S-hydroperoxide with turnover numbers of 300-400/s. AtLOX1 showed a broad range of activity over the range pH 5-9 (optimal at pH 6); tomato LOXA also showed optimal activity around pH 5-7 dropping off more sharply at pH 9. Site-directed mutagenesis of a conserved active site Ala (Ala562 in AtLOX1, Ala 564 in tomato LOXA, and typically conserved as Ala in S-LOX and Gly in R-LOX), revealed that substitution with Gly led to the production of a mixture of 9S- and 13R-hydroperoxyoctadecadienoic acids from linoleic acid. To follow up on earlier reports of 9-LOX metabolism of anandamide (van Zadelhoff et al. Biochem. Biophys. Res. Commun. 248:33-38, 1998), we also tested this substrate with the mutants, which produced predictable shifts in product profile, including a shift from the prominent 11S-hydroperoxy derivative of wild-type to include the 15R-hydroperoxide. These results conform to a model that predicts a head-first substrate binding orientation for 9S-LOX. We also found that linoleoyl-phosphatidylcholine is not a 9S-LOX substrate, which is consistent with this conclusion.
Boeglin
William E
WE
Department of Pharmacology, Vanderbilt University Medical Center, RRB Room 510, 23rd Ave at Pierce, Nashville, TN, 37232-6602, USA. alan.brash@vanderbilt.edu
Itoh
Aya
A
Zheng
Yuxiang
Y
Coffa
Gianguido
G
Howe
Gregg A
GA
Brash
Alan R
AR
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D019787:Linoleic Acid / Q000737:chemistry* / Q000378:metabolism; D008084:Lipoxygenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D018551:Solanum lycopersicum / Q000201:enzymology; D009154:Mutation; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013237:Stereoisomerism; D013379:Substrate Specificity
D010940:Plant Proteins; D019787:Linoleic Acid; D008084:Lipoxygenase; D000409:Alanine; D005998:Glycine
Can J Biochem Physiol. 1959 Aug;37(8):911-7
13671378
Biochim Biophys Acta. 1989 Feb 20;1001(3):274-81
2492826
Lipids. 1996 Aug;31(8):803-9
8869882
Biochem Biophys Res Commun. 1998 Jul 9;248(1):33-8
9675081
Methods Enzymol. 1996;272:250-9
8791784
FEBS Lett. 1975 Dec 1;60(1):149-52
6318
Proc Natl Acad Sci U S A. 2004 Nov 2;101(44):15579-84
15496467
Biochem Biophys Res Commun. 1998 Feb 13;243(2):438-43
9480827
Plant Physiol. 1993 Dec;103(4):1133-7
8290626
Lipids. 1977 Mar;12(3):324-6
403382
Biochem Biophys Res Commun. 2005 Dec 9;338(1):87-92
16111652
Annu Rev Plant Biol. 2002;53:275-97
12221977
Anal Biochem. 2000 Dec 1;287(1):186-9
11078601
Biochem Biophys Res Commun. 1985 Jul 31;130(2):559-66
3927906
Biochem Biophys Res Commun. 1992 Dec 30;189(3):1349-52
1482350
Biochim Biophys Acta. 1994 Jan 20;1210(3):297-302
7905748
Proc Natl Acad Sci U S A. 1984 Feb;81(3):689-93
6322165
Eur J Biochem. 1993 Nov 15;218(1):165-71
8243462
Plant Physiol. 2004 Sep;136(1):2641-51
15347800
Plant Physiol. 1994 Sep;106(1):109-18
7972514
Plant Physiol. 1993 Feb;101(2):441-50
7506426
Biochem Biophys Res Commun. 1972 Sep 5;48(5):1055-60
5066282
Chem Biol. 2007 May;14(5):473-88
17524979
Lipids. 1988 Apr;23(4):340-4
3398721
Arch Biochem Biophys. 1988 Sep;265(2):514-23
3138949
Methods Enzymol. 2007;433:145-57
17954233
Biochim Biophys Acta. 2001 Apr 7;1546(2):379-98
11295443
Plant Physiol. 1997 Jul;114(3):1085-93
9232884
Plant Physiol. 1994 May;105(1):385-393
12232208
Biomed Biochim Acta. 1991;50(1):11-5
1907133
Biochemistry. 1987 Aug 25;26(17):5465-71
3118947
Plant Cell. 2007 Mar;19(3):831-46
17369372
J Biol Chem. 1999 Nov 19;274(47):33764-70
10559269
Lipids. 2009 Mar;44(3):207-15
19037675
J Biol Chem. 2005 Nov 18;280(46):38756-66
16157595
Curr Opin Plant Biol. 2002 Jun;5(3):230-6
11960741