Mechanism of .alpha.-ketoglutarate oxidation in coupled enzymic oxygenations

Lindblad, Bengt; Lindstedt, Göran; Tofft, Marianne; Lindstedt, Sven

doi:10.1021/ja01044a082

Cited by 58 publications

(30 citation statements)

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“…All three mechanisms are compatible with the finding [8] that both atoms ofthe same oxygen molecule are incorporated into the product. Whereas it is difficult to differentiate experimentally between the mechanisms proposed by Witkop [3] and by Hamilton [5], the third mechanism, which involves a Diels-Alder type addition of oxygen to the enol form of 4'-hydroxyphenylpyruvate to yield structure 9, implies that one solvent proton is incorporated into the product.…”

Section: Determinution Of' the Enuntiomeric Purity Of 25-dimethoxyphsupporting

confidence: 84%

“…Several mechanisms have been suggested for this remarkable transformation [2 -71, none of them has, however, been proved. By the use of l 8 0 2 it has been shown that both atoms of molecular oxygen are incorporated into the same homogentisic acid molecule thus justifying the name dioxygenase [8].The enzyme has been isolated from mammalian, avian and human liver [9-121 and recently from Pseudoinonas sp. P.J.…”

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confidence: 99%

“…4-Hydroxyphenylpyruvate dioxygenase has been purified from beef liver to a specific activity of 0.021 U/mg at 25 "C.…”

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confidence: 99%

“…By the use of l 8 0 2 it has been shown that both atoms of molecular oxygen are incorporated into the same homogentisic acid molecule thus justifying the name dioxygenase [8].…”

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Steric Course of the NIH Shift in the Enzymic Formation of Homogentisic Acid

Leinberger

Rétey

Hull

et al. 1981

European Journal of Biochemistry

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1. 4-Hydroxyphenylpyruvate dioxygenase has been purified from beef liver to a specific activity of 0.021 U/mg at 25 "C.2. Samples of (3R)-and (35')-4'-hydro~yphenyl[3-~H~]pyruvate were prepared by taking advantage of the known stereospecificity of phenylpyruvate keto -enol-isomerase (tautomerase). These were converted in situ by the dioxygenase into the corresponding deuterium-labelled homogentisic acids, which were isolated as the crystalline 2',5'-diinethyl derivatives after treatment with dimethyl sulphate.3. 2',5'-Dimethoxy-(4-cinnamate was deuterated by enoate reductase from Clostridiurn sp. La 1 to afford (2S, 3R)-3-(2',5'-dimetho~yphenyI)-[2-~H~, propionate which was in turn chemically degraded to (S)-2,5-dimethoxyphenyl r2 Hl lacetic acid. 5. In the 500-MHz 'H-NMR spectrum the 2-methylene protons of unlabelled 2,5-dimethoxyphenylacetic acid (-)-menthy1 ester appeared as a well resolved AB system. Using a triple resonance technique and the reference samples (b) and (c) the 2-HR, atom could be correlated with the high-field half and the 2-HsI atom with the lowfield half of the AB system. 6. By comparison of the spectra of the enzymically obtained samples (d) and (e) with the reference spectra it was concluded that in the dioxygenase reaction the side-chain migration occurred with retention of configuration at the methylene C atom.4-Hydroxyphenylpyruvate dioxygenase is involved in the enzymic degradation of tyrosine and catalyses the following reaction [Eqn (l)] :The oxidative decarboxylation of the substrate is coupled with an ortho shift of the side chain reminiscent of the wellknown NIH shift of the para-hydrogen atom during the hydroxylation of phenylalanine [I, 21. Several mechanisms have been suggested for this remarkable transformation [2 -71, none of them has, however, been proved. By the use of l 8 0 2 it has been shown that both atoms of molecular oxygen are incorporated into the same homogentisic acid molecule thus justifying the name dioxygenase [8].The enzyme has been isolated from mammalian, avian and human liver [9-121 and recently from Pseudoinonas sp. P.J. 874 as well [13]. No specific prosthetic group has been yet detected. However, vitamin C and Fe2+ ions function as activators. In vim the best activator is 2,6-dichlorophenolindophenol in its reduced form [14]. (1 W) was applied over the deuterium lock circuit of the Proton magnetic 312 standard 'H-observe probe head. 'H-homodecoupling at the aromatic protons was performed simultaneously in the conventional fashion. Mass spectra were measured with a Hitachi-Perkin-Elmer RMU-6A spectrograph. The limit of error of the mass spectroscopic measurements was estimated to be 1 %. Optical rotatory dispersion (ORD) measurements were conducted with the polarimeter Jasco V. The circular dichroism (CD) curves were obtained with dichrograph Mark I11 of Jobin-Yvon. For ultraviolet spectroscopic measurements, a Unicam SP-1800 spectrophotometer was used.Dialyses were carried out with a hollow-fiber system model DC 2 of Amicon. MaterialsPhenylpyruvate ke...

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Section: Determinution Of' the Enuntiomeric Purity Of 25-dimethoxyphsupporting

confidence: 84%

mentioning

confidence: 99%

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Steric Course of the NIH Shift in the Enzymic Formation of Homogentisic Acid

Leinberger

Rétey

Hull

et al. 1981

European Journal of Biochemistry

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“…The other atom of molecular oxygen is incorporated into succinate, the decarboxylation product of 2-oxoglutarate, in a reaction typical of those catalyzed by 2-oxoglutaratedependent dioxygenases (1). Simultaneous incorporation of 1802 into a hydroxyl group and into succinate has been reported for two 2-oxoglutarate-dependent dioxygenases; y-butyrobetaine hydroxylase (20) and prolyl 4-hydroxylase (3).…”

Section: Methodsmentioning

confidence: 99%

Epoxidation in Vivo of Hyoscyamine to Scopolamine Does Not Involve a Dehydration Step

Hashimoto

Kohno²,

Yamada³

1987

Plant Physiol.

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Hyoscyamine is epoxidized to scopolamine via 6,6-hydroxyhyoscyamine in several solanaceous plants. 6,7-Dehydrohyoscyamine has been proposed to be an intermediate in the conversion of 6#-hydroxyhyoscyamine to scopolamine on the basis of the observation that this unsaturated alkaloid is converted to scopolamine when fed to a Datura scion. To determine whether a dehydration step is involved in scopolamine biosynthesis, 16-'8016j0-hydroxyhyoscyamine was prepared from I-hyoscyamine and '02 using hyoscyamine 6f6-hydroxylase obtained from root cultures of Hyoscyamus niger L. When 16-'8016,-hydroxyhyoscyamine was fed to shoot cultures of Duboisia myoporoides R. BR., the labeled alkaloid was converted to scopolamine which retained "O in the epoxide oxygen. It is concluded that 6,6-hydroxyhyoscyamine is converted in vivo to scopolamine without a dehydration step.Scopolamine, the epoxide of hyoscyamine, is one of the major alkaloids which accumulate in the family Solanaceae (7). The formation of the epoxide bridge in scopolamine begins with the hydroxylation of L-hyoscyamine to Hyos-OH' by a 2-oxoglutarate-dependent dioxygenase, hyoscyamine 6#-hydroxylase (12 De-hyos (scheme 1 in Fig. 1); '"0 should be retained if dehydration is not involved (scheme 2 in Fig. 1). MATERIALS AND METHODSChemicals. '"02 (>99 atom %) was purchased from CEA, France. L-Hyoscyamine hydrobromide and scopolamine hydrobromide were obtained from Nakarai Chemicals, Kyoto. Hyos-OH hydrobromide was prepared using hyoscyamine 6#-hydroxylase from root cultures of Hyoscyamus niger L. (12). De-hyos was synthesized by the method of Sharpless et al. (23). Other chemicals were obtained as described previously (12).Root and Shoot Cultures. Culture conditions for root cultures ofH. niger L. have been reported elesewhere (12). Shoot cultures ofDuboisia myoporoides R. BR. were initiated according to Endo et al. (6) and were subcultured on a Gyrotory shaker (model G 10-21, New Brunswick Scientific, Edison, NJ) at 100 rpm and 25°C under light (2,700-4,500 lux) in 100-ml flasks containing 25 ml of liquid B5 medium (10) supplemented with 3% (w/v) sucrose and 10 Mm 6-BA. In the feeding experiments, alkaloid solutions were neutralized with HCI when necessary and were sterilized by passing through 0.22 Am membrane filters. These were added to 10 ml ofthe above culture medium in 50-ml flasks to a final concentration of0.2 mm. The shoot cultures containing young leaves were inoculated in each flask and cultured under the above conditions.Partial Purification of Hyoscyamine 6,8-Hydroxylase. Hyoscyamine 6t3-hydroxylase was partially purified from root cultures ofH. niger. The hydroxylase in the crude extract was precipitated between 60 and 80% saturation of (NH4)2SO4 and subsequently chromatographed on a DEAE-Toyopearl 650M column. The detailed purification procedure has been reported (13). The partially purified enzyme preparation (about 23-fold in specific activity) was concentrated using Amicon

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