Hydroxymellein synthase

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... 

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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