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A Determinant of Substrate Specificity Predicted from the
Acyl-Acyl Carrier Protein Desaturase of Developing
Cat's Claw
Seed1
Edgar B. Cahoon2, 3,
Salehuzzaman Shah2, 4,
John Shanklin, and
John Browse*
Biology Department, Brookhaven National Laboratory, Upton, New York
11976 (E.B.C., J.S.); and Institute of Biological Chemistry,
Washington State University, P.O. Box 646340, Pullman, Washington
99164-6340 (S.S., J.B.)
Cat's
claw (Doxantha unguis-cati L.) vine accumulates nearly
80% palmitoleic acid (16:1 9) plus cis-vaccenic acid
(18:111) in its seed oil. To characterize the biosynthetic origin of
these unusual fatty acids, cDNAs for acyl-acyl carrier protein
(acyl-ACP) desaturases were isolated from developing cat's claw seeds.
The predominant acyl-ACP desaturase cDNA identified encoded a
polypeptide that is closely related to the stearoyl (9-18:0)-ACP
desaturase from castor (Ricinis communis
L.) and other species. Upon expression in
Escherichia coli, the cat's claw polypeptide functioned
as a 9 acyl-ACP desaturase but displayed a distinct substrate
specificity for palmitate (16:0)-ACP rather than stearate (18:0)-ACP.
Comparison of the predicted amino acid sequence of the cat's claw
enzyme with that of the castor 9-18:0-ACP desaturase suggested that a single amino acid substitution (L118W) might account in large part
for the differences in substrate specificity between the two
desaturases. Consistent with this prediction, conversion of leucine-118
to tryptophan in the mature castor 9-18:0-ACP desaturase resulted
in an 80-fold increase in the relative specificity of this enzyme for
16:0-ACP. The alteration in substrate specificity observed in the L118W
mutant is in agreement with a crystallographic model of the proposed
substrate-binding pocket of the castor 9-18:0-ACP desaturase.
1
This work was supported by the U.S. Department
of Agriculture-National Research Initiative Competitive Grants Program
(grant no. 97-35301-4426 to J.B.), the Office of Basic Energy Sciences of the U.S. Department of Energy (E.C., J.S.), and the Agricultural Research Center, Washington State University.
2
These authors contributed equally to the work
and are considered joint first authors.
3
Present address: DuPont Agricultural Products,
Experimental Station, Building 402, Wilmington, DE 19880.
4
Present address: Alberta Research Council, P.O.
Box 4000, Vegreville, Alberta T9C 1T4, Canada.
*
Corresponding author; e-mail jab{at}wsu.edu; fax 1-509-335-7643.
Plant Physiol. (1998) 117: 593-598
Copyright Clearance Center: 0032-0889/98/117/0593/06
© 1998 American Society of Plant Physiologists
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