Functional characterization of premnaspirodiene oxygenase, a cytochrome P450 catalyzing regio- and stereo-specific hydroxylations of diverse sesquiterpene substrates.
Solavetivone, a potent antifungal phytoalexin, is derived from a vetispirane-type sesquiterpene, premnaspirodiene, by a putative regio- and stereo-specific hydroxylation, followed by a second oxidation to yield the alpha,beta-unsaturated ketone. Mechanistically, these reactions could occur via a single, multifunctional cytochrome P450 or some combination of cytochrome P450s and a dehydrogenase. We report here the characterization of a single cytochrome P450 enzyme, Hyoscyamus muticus premnaspirodiene oxygenase (HPO), that catalyzes these successive reactions at carbon 2 (C-2) of the spirane substrate. HPO also catalyzes the equivalent regio-specific (C-2) hydroxylation of several eremophilane-type (decalin ring system) sesquiterpenes, such as with 5-epi-aristolochene. Moreover, HPO displays interesting comparisons to other sesquiterpene hydroxylases. 5-Epi-aristolochene di-hydroxylase (EAH) differs catalytically from HPO by introducing hydroxyl groups first at C-1, then C-3 of 5-epi-aristolochene. HPO and EAH also differ from one another by 91-amino acid differences, with four of these differences mapping to putative substrate recognition regions 5 and 6. These four positions were mutagenized alone and in various combinations in both HPO and EAH and the mutant enzymes were characterized for changes in substrate selectivity, reaction product specificity, and kinetic properties. These mutations did not alter the regio- or stereo-specificity of either HPO or EAH, but specific combinations of the mutations did improve the catalytic efficiencies 10-15-fold. Molecular models and comparisons between HPO and EAH provide insights into the catalytic properties of these enzymes of specialized metabolism in plants.
Takahashi
Shunji
S
Plant Biology Program, Department of Plant and Soil Sciences, University of Kentucky, Lexington, Kentucky 40546-0312, USA.
Yeo
Yun-Soo
YS
Zhao
Yuxin
Y
O'Maille
Paul E
PE
Greenhagen
Bryan T
BT
Noel
Joseph P
JP
Coates
Robert M
RM
Chappell
Joe
J
D000595:Amino Acid Sequence; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D018744:DNA, Plant / Q000737:chemistry; D006900:Hydroxylation; D032304:Hyoscyamus / Q000201:enzymology; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010105:Oxygenases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D018744:DNA, Plant; D012717:Sesquiterpenes; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases
Nat Biotechnol. 2002 Nov;20(11):1135-9
12368811
Arch Biochem Biophys. 2001 Sep 15;393(2):222-35
11556809
Appl Microbiol Biotechnol. 2005 Jun;67(4):477-83
15602686
Trends Plant Sci. 2004 Sep;9(9):433-40
15337493
J Biol Chem. 1997 Aug 1;272(31):19176-86
9235908
Plant Physiol. 1976 May;57(5):760-5
16659566
Plant Physiol. 2001 Apr;125(4):1930-40
11299372
J Biol Chem. 1964 Jul;239:2370-8
14209971
Chem Res Toxicol. 1996 Jul-Aug;9(5):871-4
8828923
J Biol Chem. 1992 Jan 5;267(1):83-90
1730627
Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):11088-92
1438319
Nat Biotechnol. 2001 Apr;19(4):371-4
11283597
Arch Biochem Biophys. 2003 Jan 15;409(2):385-94
12504906
Life Sci. 2005 Dec 22;78(5):431-41
16198377
J Nat Prod. 1998 Dec;61(12):1516-9
9868154
Arch Biochem Biophys. 1999 Aug 1;368(1):181-92
10415126
J Am Chem Soc. 2003 Jan 22;125(3):705-14
12526670
Proc Natl Acad Sci U S A. 2006 Jun 27;103(26):9826-31
16785438
Arch Biochem Biophys. 2004 Feb 1;422(1):31-41
14725855
Arch Biochem Biophys. 2004 Jul 15;427(2):143-53
15196988
Methods Enzymol. 2003;374:461-91
14696385
Plant Cell. 1994 Sep;6(9):1191-1192
12244269
Proteins. 2003 Sep 1;52(4):609-23
12910460
Planta. 2005 Jul;221(5):603-6
15931500
DNA Cell Biol. 1993 Jan-Feb;12(1):1-51
7678494
Biochemistry. 2004 Feb 3;43(4):981-8
14744142
J Biol Chem. 1995 Mar 31;270(13):7375-81
7706281
Biosci Biotechnol Biochem. 2004 Dec;68(12):2640-2
15618642
J Biol Chem. 2005 Feb 4;280(5):3686-96
15522862
J Biol Chem. 2004 Jun 25;279(26):27294-301
15100217
J Biol Chem. 2005 May 20;280(20):19569-75
15774478
Plant Physiol. 1988 Dec;88(4):1291-6
16666457
J Org Chem. 2004 Oct 29;69(22):7428-35
15497966
Mycorrhiza. 2003 Dec;13(6):333-6
14505123
Proc Natl Acad Sci U S A. 1998 Aug 18;95(17):9903-8
9707573