A multifunctional acyl-acyl carrier protein desaturase from Hedera helix L. (English ivy) can synthesize 16- and 18-carbon monoene and diene products.
A desaturase with 83% sequence identity to the coriander delta(4)-16:0-ACP desaturase was isolated from developing seeds of Hedera helix (English ivy). Expression of the ivy desaturase in Arabidopsis resulted in the accumulation of 16:1delta(4) and its expected elongation product 18:1delta(6) (petroselinic acid). Expression in Escherichia coli resulted in the accumulation of soluble, active protein that was purified to apparent homogeneity. In vitro assays confirmed delta(4) desaturation with 16:0-ACP; however, with 18:0-acyl acyl carrier protein (ACP) desaturation occurred at the delta(9) position. The ivy desaturase also converted 16:1delta(9)-ACP and 18:1delta(9)-ACP to the corresponding delta(4,9) dienes. These data suggest at least two distinct substrate binding modes, one placing C4 at the diiron active site and the other placing C9 at the active site. In the latter case, 18:0 would likely bind in an extended conformation as described for the castor desaturase with 9-carbons accommodated in the cavity beyond the dirron site. However, delta(4) desaturation would require the accommodation of 12 carbons for C16 substrates or 14 carbons for C18 substrates. The amino acids lining the substrate binding cavity of ivy and castor desaturases are conserved except for T117R and P179I (castor/ivy). Paradoxically, both substitutions, when introduced into the castor desaturase, favored the binding of shorter acyl chains. Thus, it seems likely that delta(4) desaturation would require a non-extended, perhaps U-shaped, substrate conformation. A cis double bond may facilitate the initiation of such a non-extended conformation in the monounsaturated substrates. The multifunctional properties of the ivy desaturase make it well suited for further dissection of the determinants of regiospecificity.
Whittle
Edward
E
Biology Department, Brookhaven National Laboratory, Upton, New York, 11973, USA.
Cahoon
Edgar B
EB
Subrahmanyam
Satyam
S
Shanklin
John
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D028526:Coriandrum / Q000201:enzymology; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology; D035981:Hedera / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009829:Oleic Acids / Q000096:biosynthesis*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D009829:Oleic Acids; C008820:petroselinic acid; D006899:Mixed Function Oxygenases; C018747:acyl-(acyl-carrier-protein)desaturase