title: Revealing the catalytic potential of an acyl-ACP desaturase: tandem selective oxidation of saturated fatty acids.
abstract: It is estimated that plants contain thousands of fatty acid structures, many of which arise by the action of membrane-bound desaturases and desaturase-like enzymes. The details of "unusual" e.g., hydroxyl or conjugated, fatty acid formation remain elusive, because these enzymes await structural characterization. However, soluble plant acyl-ACP (acyl carrier protein) desaturases have been studied in far greater detail but typically only catalyze desaturation (dehydrogenation) reactions. We describe a mutant of the castor acyl-ACP desaturase (T117R/G188L/D280K) that converts stearoyl-ACP into the allylic alcohol trans-isomer (E)-10-18:1-9-OH via a cis isomer (Z)-9-18:1 intermediate. The use of regiospecifically deuterated substrates shows that the conversion of (Z)-9-18:1 substrate to (E)-10-18:1-9-OH product proceeds via hydrogen abstraction at C-11 and highly regioselective hydroxylation (>97%) at C-9. (18)O-labeling studies show that the hydroxyl oxygen in the reaction product is exclusively derived from molecular oxygen. The mutant enzyme converts (E)-9-18:1-ACP into two major products, (Z)-10-18:1-9-OH and the conjugated linolenic acid isomer, (E)-9-(Z)-11-18:2. The observed product profiles can be rationalized by differences in substrate binding as dictated by the curvature of substrate channel at the active site. That three amino acid substitutions, remote from the diiron active site, expand the range of reaction outcomes to mimic some of those associated with the membrane-bound desaturase family underscores the latent potential of O(2)-dependent nonheme diiron enzymes to mediate a diversity of functionalization chemistry. In summary, this study contributes detailed mechanistic insights into factors that govern the highly selective production of unusual fatty acids.
authors.affiliation: Department of Biology, Brookhaven National Laboratory, 50 Bell Avenue, Upton, NY 11973, USA.
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Whittle
authors.forename: Amy E
authors.initials: AE
authors.lastname: Tremblay
authors.forename: Peter H
authors.initials: PH
authors.lastname: Buist
authors.forename: John
authors.initials: J
authors.lastname: Shanklin
mesh_terms: D001665:Binding Sites; D005227:Fatty Acids / Q000378:metabolism*; D006900:Hydroxylation; D007536:Isomerism; D007700:Kinetics; D019787:Linoleic Acid / Q000737:chemistry; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D020005:Propanols / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012278:Ricinus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D005227:Fatty Acids; D020005:Propanols; C006463:allyl alcohol; D019787:Linoleic Acid; D006899:Mixed Function Oxygenases; C018747:acyl-(acyl-carrier-protein)desaturase; D010100:Oxygen
references.citation: Biochemistry. 2003 Jul 1;42(25):7828-35
references.pmid: 12820892
references.citation: Nat Prod Rep. 2004 Apr;21(2):249-62
references.pmid: 15042148
references.citation: J Biol Chem. 2003 Jul 4;278(27):25072-80
references.pmid: 12704186
references.citation: Biochem Biophys Res Commun. 2005 Dec 9;338(1):240-9
references.pmid: 16168953
references.citation: Gene. 1989 Apr 15;77(1):51-9
references.pmid: 2744487
references.citation: Proc Natl Acad Sci U S A. 2000 Oct 24;97(22):12350-5
references.pmid: 11027301
references.citation: Proc Natl Acad Sci U S A. 1999 Oct 26;96(22):12935-40
references.pmid: 10536026
references.citation: Annu Rev Plant Physiol Plant Mol Biol. 1998 Jun;49:611-641
references.pmid: 15012248
references.citation: Prog Lipid Res. 1983;22(4):235-56
references.pmid: 6359174
references.citation: J Biol Chem. 2001 Jun 15;276(24):21500-5
references.pmid: 11294879
references.citation: J Am Chem Soc. 2002 Apr 3;124(13):3277-83
references.pmid: 11916411
references.citation: EMBO J. 1996 Aug 15;15(16):4081-92
references.pmid: 8861937
references.citation: Proc Natl Acad Sci U S A. 1997 Apr 1;94(7):2981-6
references.pmid: 9096332
references.citation: Trends Plant Sci. 2000 Mar;5(3):95-101
references.pmid: 10707074
references.citation: Lipids. 1975 Apr;10(4):215-9
references.pmid: 27521068
references.citation: Science. 1998 May 8;280(5365):915-8
references.pmid: 9572738
references.citation: J Inorg Biochem. 2000 Jan 15;78(1):7-14
references.pmid: 10714700
references.citation: J Biol Chem. 2007 Jul 6;282(27):19863-71
references.pmid: 17463003
references.citation: Science. 1998 Nov 13;282(5392):1315-7
references.pmid: 9812895
references.citation: Proc Natl Acad Sci U S A. 2007 Oct 16;104(42):16444-9
references.pmid: 17921252
references.citation: Protein Sci. 2005 Jun;14(6):1508-17
references.pmid: 15929999
references.citation: J Biol Chem. 2002 May 3;277(18):15613-20
references.pmid: 11864983
references.citation: Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2486-90
references.pmid: 8460163
references.citation: Prostaglandins Leukot Essent Fatty Acids. 2003 Feb;68(2):73-95
references.pmid: 12538072
references.citation: Biochemistry. 2002 Aug 6;41(31):10141-8
references.pmid: 12146979
references.citation: Chem Commun (Camb). 2004 Nov 7;(21):2406-7
references.pmid: 15514788
references.citation: J Biol Chem. 1960 Feb;235:337-45
references.pmid: 13801633
references.citation: Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2510-4
references.pmid: 2006187
references.citation: Proc Natl Acad Sci U S A. 1995 Jul 18;92(15):6743-7
references.pmid: 7624314
references.citation: J Biol Chem. 2004 Mar 26;279(13):12495-502
references.pmid: 14718523
references.citation: Acc Chem Res. 2004 Jul;37(7):421-9
references.pmid: 15260504