Revealing the catalytic potential of an acyl-ACP
desaturase: Tandem selective oxidation of
saturated fatty acids
Edward J. Whittle*, Amy E. Tremblay†, Peter H. Buist,†and John Shanklin*‡
*Department of Biology, Brookhaven National Laboratory, 50 Bell Avenue, Upton, NY 11973; and†Department of Chemistry, Carleton University,
1125 Colonel By Drive, Ottawa, ON, Canada K1S 5B6
Edited by Wendell Roelofs, Cornell University, Geneva, NY, and approved July 14, 2008 (received for review June 10, 2008)
It is estimated that plants contain thousands of fatty acid struc-
tures, 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. How-
ever, 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 hydroxy-
lation (>97%) at C-9.18O-labeling studies show that the hydroxyl
oxygen in the reaction product is exclusively derived from molec-
ular 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
the membrane-bound desaturase family underscores the latent
potential of O2-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.
binuclear iron ? diiron ? hydroxylation ? nonheme iron ? catalysis
fication reactions are initiated by hydrogen removal from an
unactivated methylene/vinyl group and result in oxidation of
substrate (2). In all reactions described to date, an activated
oxygen species formed at a diiron center (3, 4) is believed to
effect the initial hydrogen abstraction. The recognition of this
coworkers (5) to identify the gene encoding the oleoyl 12-
hydroxylase based on its homology to the oleoyl 12-desaturase.
Appreciation of the catalytic diversity of desaturase-like en-
zymes (6) stimulated a search for, and subsequent identification
of, an acetylenase, epoxygenase (7), and enzymes responsible for
the synthesis of conjugated fatty acids (8) and allylic alcohols (9).
Progress toward understanding the determinants of reaction
outcome has been achieved via comparative studies on the
oleate-desaturase/hydroxylase pair involving systematic site-
directed mutagenesis of residues proximal to the putative active
site (10, 11). Unfortunately, further interpretation of these data
will not be possible until this class of membrane-bound enzymes
is structurally characterized.
Several crystal structures (12–15) are available for the struc-
turally distinct, soluble acyl-acyl carrier protein (ACP) desatu-
t has been estimated that the seeds of plants contain thousands
of structurally distinct fatty acids (1). Many fatty acid modi-
rases (6, 16–18) but this class of enzyme acts only as a desaturase
with natural substrates and precludes the desired correlation of
structure with a range of reaction outcomes. The acyl-ACP
family of desaturases contains a number of members with
different substrate chain length specificities and regioselectivi-
ties (6, 19). However, the absence of reported oxygenation
striking. In the present work, we describe a triple mutant of the
castor desaturase that is able to convert stearoyl- or oleoyl-ACP
to the allylic alcohol (E)-10-18:1-9-OH with high regioselectiv-
ity; the (E)-isomer of oleoyl-ACP, elaidyl-ACP, is converted to
a mixture of (Z)-10-18:1-9-OH and the conjugated 9-(E)-11-(Z)
isomer of linoleic acid (CLA). The different reaction outcomes
are rationalized by distinct substrate binding modes in a curved
Results and Discussion
Mutant Desaturase Converts Stearoyl Substrate to a Hydroxylated
Product. During the course of experiments designed to under-
stand the factors governing regioselectivity, we engineered a
triple mutant T117R/G188L/D280K of the castor ?9 desaturase
(Fig. 1) (referred to in this article as ‘‘mutant desaturase’’) in an
attempt to mimic structural features of the ivy ?4 desaturase
(14). The expected change in regioselectivity from ?9 to ?4 was
not observed; however, the 18:0 substrate was converted into
(Z)-9-18:1 product, which initially accumulated and then disap-
peared, as indicated by GC analysis (Fig. 2 A–C). The apparent
mass balance deficit was restored upon derivatization of the
product mixture with (N,O-bis[Trimethylsily]trifluoroac-
etamide) plus trimethylchlorosilane, whereupon a ?9:1 mixture
of two new products (Fig. 2D) was observed. This result sug-
gested that these novel products were alcohols. The mass spectra
of the trimethylsilyl (TMS) derivatives of the major and minor
hydroxylated product (Fig. 2 E and F) were indistinguishable
from each other and featured an apparent molecular ion (m/z ?
384) consistent with a C18 product containing an O-TMS group
and a double bond. The presence of an m/z 227 ion in these mass
spectra is diagnostic for an O-TMS group at C-9 and a double
bond between C-9 and the methyl group of the fatty acid. The
initial formation of (Z)-9-18:1-ACP, and its subsequent decrease
in abundance with hydroxy product accumulation indicated that
(Z)-9-18:1-ACP was a substrate for the mutant enzyme. This
interpretation was confirmed by the observation of hydroxylated
Author contributions: J.S. designed research; E.J.W., A.E.T., and J.S. performed research;
A.E.T. and P.H.B. contributed new reagents/analytic tools; E.J.W., A.E.T., P.H.B., and J.S.
analyzed data; and P.H.B. and J.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
‡To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
September 23, 2008 ?
vol. 105 ?
reaction was terminated by the introduction of toluene esterified and sily-
lated as described previously.
ACKNOWLEDGMENTS. E.J.W. and J.S. thank the Office of Basic Energy Sci-
ences of the U.S. Department of Energy, for financial support of this work.
Support was also provided by Natural Sciences and Engineering Research
Council (NSERC) (CGS-M postgraduate scholarship program to A.E.T., Grant
OGP-2528 to P.H.B.). We thank Pat Covello for providing some of the deuter-
ated compounds and Jodie Guy for structural comparisons of ivy and castor
desaturase and for graphics assistance.
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