Induction and Specificity of a (Cytochrome P-450)-Dependent Laurate In-chain-Hydroxylase from Higher Plant Microsomes

Abstract

The substrate and product specifities of the (cytochrome P-450)-dependent laurate monooxygenase from tuber tissues of Jerusalem artichoke (Helianthus tuberosus L.) were investigated. The plant enzyme appeared strictly specific for the Clz free fatty acid and produced a mixture of C-8, C-9 and C-10 hydroxylated lauric acids, the C-9 derivative being predominant. No (2-12 or C-11 hydroxylated laurates were detected. The activity of the enzyme, which was not detectable in the intact tuber, was induced by slicing and aging the tissues on water, and strongly superinduced by the addition of manganese and phenobarbital to the aging medium. Regulation of laurate hydroxylase was clearly independent from that of cinnamic acid 4-hydroxylase, another plant cytochrome P-450 enzyme.

Cytochrome P-450-Dependent ω-Hydroxylation of Lauric Acid by Microsomes from Pea Seedlings

“…Lauric acid w-hydroxylation was assayed by a procedure modified from Salaun et al (15). In a final volume of 2 ml, the reaction mixture contained: 1 mM NADPH, 6.7 mM glucose 6-P, 0.4 units glucose 6-P dehydrogenase, [1-14C] CYT P-450-DEPENDENT o-HYDROXYLATION Cinnamic acid 4-hydroxylation was measured with the radiochemical method described previously (2).…”

“…feeding to the tissues metabolic intermediates, the induction of this enzyme could be clearly dissociated from that of the cinnamic acid 4-hydroxylase, thus providing some evidence that the two monooxygenase activities were supported by distinct hemoproteins (15,16). In this paper, we describe a different lauric acid monooxygenase in pea seedlings.…”

“…We have recently reported that aging Jerusalem artichoke tuber tissues contain an in-chain hydroxylating lauric acid monooxygenase (14) which, unlike the mammalian and microbial fatty acid hydroxylases, showed very strict substrate specificity (15). By ' Supported by the Centre National de la Recherche Scientifique, Equipe de Recherche Associ&e 104 and Grant ATP 4117.…”

Molecular Analysis and Heterologous Expression of an Inducible Cytochrome P-450 Protein from Periwinkle (Catharanthus roseus L.)

“…For some experiments, the parsley cell cultures were induced by irradiation with UV-A (7). The Jerusalem artichoke (Helianthus tuberosus L.) system has been described (21). Parsley leaves were purchased at a local market.…”

“…The measurements of CA4H (7), LAH (21), and NADPH Cyt c reductase (6) The library was sequentially screened in two steps to obtain a subset of clones corresponding to induced mRNAs of the sizes expected for P-450 proteins: (a) selection of clones representing induced mRNAs (differential hybridization with labeled single-stranded cDNAs from noninduced and induced cultures), and (b) selection of a second subset of cDNAs that hybridize in northem blots to induced mRNAs of the size expected for P-450 transcripts (1.3-1.9 kb for proteins of 45 to 65 kD; the known eukaryotic P-450 enzymes are in this size group [19]). The techniques have been described (22,24 The open reading frame in cDNA pCrosl was fused in frame with the amino terminal part of MS2 polymerase in expression vector pEX31c.…”