Effect of phosphate residue of NADPH on the interaction between catalytic domains of a multifunctional polyketide synthetic enzyme 6-hydroxymellein synthase

AbstractAssociation of NADPH with the ketoreducing domain of 6-hydroxymellein synthase, a multifunctional polyketide synthetic enzyme of carrot, evoked the alternation of microstructure around the primary binding site of the co-substrates acetyl- and malonyl-CoAs, and this resulted in the marked decrease in Km value of the enzyme protein for acetyl-CoA. In contrast, the enzyme did not show the increase in the affinity to the substrate when NADPH was replaced by NADH. These results suggest that the phosphate residue attached to 2′-position of adenosyl moiety of NADPH molecule plays an important role in the co-operative interaction between these functional domains of the synthase

レンギョウ属(モクセイ科) の染色体数

モクセイ科レンギョウ属には6 種または7 種が知られている。そのうちの1 種Forsythia europaeaだけがヨーロッパに自生する。わが国には，中国原産のレンギョウ（F. suspensa），シナレンギョウ（F. viridissima var. viridissima），チョウセンレンギョウ（F. viridissima var. koreana）が薬用および花木として栽培され，ヤマトレンギョウ（F. japonica var. japonica）とショウドシマレンギョウ（F. togashii）の2 種が自生している。レンギョウ，チョウセンレンギョウ，ヤマトレンギョウ，ショウドシマレンギョウの4 分類群について染色体数を調べた結果，いずれも2n=28 であった。レンギョウとチョウセンレンギョウでは過去の報告と一致し，ヤマトレンギョウとショウドシマレンギョウでは染色体数が初めて明らかになった。シナレンギョウ，F. europaea, F. giraldiana, F.×intermedia およびF. japonica var. saxatilis もすべて2n=28 であることが知られている。レンギョウ属の染色体基本数はx=14（Taylor 1945）であることから，この属はすべて二倍体（2n=28）あることが判った

Transacylase-like structure and its role in substrate channeling of 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme in carrot cell extracts

Abstract6-Hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme of carrot, lost the binding ability toward its co-substrates, acetyl- and malonyl-CoAs, by the treatment with the blocking reagents for serine-OH. In contrast, the enzyme retained the binding ability even when the two SH groups at the reaction center (cysteine-SH of the condensation enzyme and cysteamine-SH of acyl carrier protein) were blocked, and one substrate bound to the SH-blocked enzyme was readily replaced by the other. It appeared that the cysteine-SH accepted only acetyl moiety while cysteamine-SH was preferentially malonylated in the presence of both of the substrates. These results suggest that transacylase-like domain is involved in the structure of 6-hydroxymellein synthase as a common primary binding site of its co-substrates, and acetyl and malonyl moieties are properly channeled from their CoA esters to cysteine-SH and acyl carrier protein-SH via this domain, respectively

Role of reducing co-factors in catalytic reactions of 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme in carrot cells

Abstract 6-Hydroxymellein (6HM) synthase, a multifunctional polyketide biosynthetic enzyme in carrot cells, is capable of catalyzing the acyl-CoA condensation and the ketoreduction in the presence of the nucleotide reducing co-factors. Although free CoA at high concentrations functioned as the activator of the NADPH-dependent 6HM formation, the compound exhibited an appreciable inhibitory activity toward the reaction mediated by NADH. CoA showed a potent inhibitory activity against substrate entry into the reaction center of the NADH-associated enzyme while, in the presence of NADPH, the compound slightly inhibited the formation of the acylated enzyme. The catalytic rate of the synthase was appreciably decreased when NADPH was replaced by the deuterium-labeled compound, however, the kH/kD value was markedly reduced if NADH and [D]NADH were employed as the reducing co-factors. These results suggest that the phosphate group attached to 2'-position of the adenosyl moiety of NADPH associated with the ketoreducing domain of 6HM synthase plays an important role in the regulation of the enzyme activity