7- Hydroxymellein

KUROSAKI, F KIZAWA, Y., NISHI, A., Derailment product in NADPH-dependent synthesis of a dihydroisocoumarin 6-hydroxymellein by elicitor-treated carrot cell extracts, Eur. J. Biochem., 1989,185, 85-89. 

Based on 13C-NMR analyses, it has been assumed [62] that 6-methoxymellein, a polyketide compound, is synthesized by head-to-tail condensation of one acetyl-CoA and four malonyl-CoA to form 3,4-dehydro-6-hydroxymellein as an intermediate. This isocoumarin derivative is then reduced to its dihydro-form, 6-hydroxymellein, which then accepts a methyl-unit via Omethyltransferase. We have shown [63] that cell extracts prepared from elicitor-treated carrot root disks catalyzes... 

Interaction between catalytic domains of 6-hydroxymellein synthase... 

When the catalytic reaction of 6-hydroxymellein synthase is carried out in the absence of NADPH or with monomeric enzyme, keto-reduction of the carbonyl group of the triketomethylene chain does not take place, and the synthase liberates triacetic acid lactone instead of 6-hydroxymellein [64, 71]. However, the efficiencies of product formation are markedly lower than for the normal reaction. Two mechanisms could account for the low efficiency of triacetic acid lactone formation observed in the monomeric and the NADPH-depleted dimeric forms of 6-hydroxymellein synthase. These are 1) Reduced affinity of the cosubstrates acetyl-CoA and/or malonyl-CoA for the enzyme protein with the incomplete reaction centers 2) Reduced rate of reaction of acyl-CoA condensation and/or product liberation. To examine these possibilities, kinetic parameters of the two triacetic acid lactone-forming reactions were compared with those of the normal reaction which produces 6-hydroxymellein. The Km value of 6-hydroxymellein synthase for acetyl-CoA in the normal reaction was estimated to be 22 pM, while in both the NADPH-depleted dimer and the monomer reactions the affinity of 6-hydroxymellein synthase protein for acetyl-CoA was markedly lower at 284 and 318 pM respectively. By contrast the Km values for malonyl-CoA in the normal and the two abnormal reactions were essentially the same (40 - 43 pM), indicating that the affinity of 6-hydroxymellein... 

Hydroxymellein production by the synthase was also observed when NADH was employed instead of NADPH. However, the effect of NADPH on enzyme activity was not fully replicated by NADH, and the activity of NADH-dependent 6-hydroxymellein production of the synthase was usually 50 - 70% of that for the corresponding NADPH-mediated reaction [80]. To clarify the biochemistry of the lower yield using NADH, the kinetic parameters of the reactions were determined under various reaction conditions. The Km value of 6-hydroxymellein synthase for NADPH was estimated to be 70 pM, while for NADH it was 10 pM, so that the affinity of the enzyme protein for NADPH is appreciably lower than for NADH. It follows that the difference in affinities of NADPH and NADH for the enzyme protein is not responsible for the low efficiency of NADH-mediated product formation. 

Initial processes of 6-hydroxymellein synthase-catalyzing reactions... 

Since preliminary studies showed that 6-hydroxymellein-O-methyl-transferase activity was appreciably inhibited in the presence of the reaction products, the mode of product inhibition of the enzyme was studied in detail in order to understand the regulatory mechanism of in vivo methyltransfer. It is well known that S-adenosyl-Z.-homocysteine (SAH), which is a common product of many O-methyltransferases that use SAM as methyl donor, is usually a potent inhibitor of such enzymes. In the 6-hydroxymellein-Omethyltransferase catalyzing reaction another product of this enzyme, 6-methoxymellein, has pronounced inhibitory activity, in addition to SAH. Since the specific product of the transferase reaction, 6-methoxymellein, is capable of inhibiting transferase activity [88], this observation suggests that activity of the transferase is specifically regulated in response to increases in cellular concentrations of its reaction products in carrot cells. It has been also found that 6-methoxymellein inhibits transferase activity with respect not only to 6-hydroxymellein but also to SAM, competitively. This competitive inhibition was also found in SAH as a function of the co-substrates of the enzyme [89]. It follows that the reaction catalyzed by 6-hydroxymellein-O-methyltransferase proceeds by a sequential bireactant mechanism in which the entry of the co-substrates to form the enzyme-substrate complexes and the release of the co-products to generate free enzyme take place in random order [Fig. (7)]. This result also implies that 6-methoxymellein and SAH have to associate with the free transferase protein to exhibit their inhibitory activities, and cannot work as the inhibitors after the enzyme forms complexes with the the substrate. If, therefore, 6-hydroxymellein-O-methyltransferase activity is controlled in vivo by its specific product 6-methoxymellein, this compound should... 

Fig. (7). Mechanisms of substrate entry and product release of 6-hydroxymellein-O-methy I transferase...

In carrot, Daucus carota (Umbelliferae), 6-methoxymellein was identified as a phytoalexin (Figure 29). It is biosynthesized by 6-hydroxymellein-O-methyltransferase from 6-hydroxymellein, which is biosynthesized from 1 mol of acetyl coenzyme A (CoA) and 4 mol of malonyl-CoA by a polyketide biosynthetic enzyme, 6-hydroxymellein hydroxylase.2 ... 

The presumed biosynthetic precursor of 6-methoxymellein, 6-hydroxymellein (10), has also been isolated from carrot roots stored under stress (ref.32). This compound was first isolated from a mutant of Aspergillus terreus (ref.42) and subsequently has been found in the bar)c of Tabebuia avellanedae (ref. 43) and produced by the fungi Pyricularla oryzae (ref. 44) and Ceratocystis minor (ref.45). Recently, the ( + )-6-hydroxymellein isomer has been isolated from the flowers of Cassia siamea (ref.46) and from a fungus of the genus Discula (ref.47). 

Kurosaki, F. (1996) Effect of NADPH associated keto-reducing domain on substrate entry into 6-hydroxymellein synthase, a multifunctional polyketide synthetic enzyme involved in phytoalexin biosynthesis in carrot Arch. Biochem. Biophys. 328, 213-217... 

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