- Investigation of the substrate range of CYP199A4: Modification of the partition between hydroxylation and desaturation activities by substrate and protein engineering
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The cytochrome P450 enzyme CYP199A4, from Rhodopseudomonas palustris HaA2, can efficiently demethylate 4-methoxybenzoic acid. It is also capable of oxidising a range of other related substrates. By investigating substrates with different substituents and ring systems we have been able to show that the carboxylate group and the nature of the ring system and the substituent are all important for optimal substrate binding and activity. The structures of the veratric acid, 2-naphthoic acid and indole-6-carboxylic acid substrate-bound CYP199A4 complexes reveal the substrate binding modes and the side-chain conformational changes of the active site residues to accommodate these larger substrates. They also provide a rationale for the selectivity of product oxidation. The oxidation of alkyl substituted benzoic acids by CYP199A4 is more complex, with desaturation reactions competing with hydroxylation activity. The structure of 4-ethylbenzoic acid-bound CYP199A4 revealed that the substrate is held in a similar position to 4-methoxybenzoic acid, and that the C β C-H bonds of the ethyl group are closer to the heme iron than those of the Cα (3.5 vs. 4.8 A?). This observation, when coupled to the relative energies of the reaction intermediates, indicates that the positioning of the alkyl group relative to the heme iron may be critical in determining the amount of desaturation that is observed. By mutating a single residue in the active site of CYP199A4 (Phe185) we were able to convert the enzyme into a 4-ethylbenzoic acid desaturase. Engineering a P450 desaturase: The substrate range of CYP199A4 from Rhodopseudomonas palustris was investigated. The partition between the hydroxylation and desaturation activities of 4-ethylbenzoic acid was studied by changing the substrate and by mutation. The activity of CYP199A4 with 4-ethylbenzoic acid was changed to a desaturase by a single mutation at F185. Copyright
- Bell, Stephen G.,Zhou, Ruimin,Yang, Wen,Tan, Adrian B. H.,Gentleman, Alexander S.,Wong, Luet-Lok,Zhou, Weihong
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p. 16677 - 16688
(2013/03/28)
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- Characterization of phenolic compounds in rooibos tea
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Polyphenols present in rooibos, a popular herbal tea from Aspalathus linearis, were isolated in two steps. First, phenolic ingredients were separated by multilayer countercurrent chromatography (MLCCC). Preparative high-performance liquid chromatography (HPLC) was then applied to obtain pure flavonoids. The purity and identity of isolated compounds was confirmed by different NMR experiments, HPLC-diode array detector (DAD), or gas chromatography-mass spectrometry (GC-MS) analysis. This strategy proved to be valid to isolate material in up to gram quantities and to verify known and previously not published polyphenol structures. In addition the chemistry of dihydrochalcones and related intermediates was studied. The dihydrochalcone aspalathin was oxidized to the corresponding flavanone-C-glycosides ((R)/(S)-eriodictyol-6-C-β-D-glucopyranoside and (R)/(S)-eriodictyol-8-C- β-D-glucopyranoside). Flavanone-6-C-β-D-glucopyranosides were further degraded to flavones isoorientin and orientin.
- Krafczyk, Nicole,Glomb, Marcus A.
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p. 3368 - 3376
(2008/09/20)
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- Metabolic pathways of 4-[(3-methoxyphenyl)methyl]-2,2,6,6-tetramethyl-1-oxa-4-aza-2,6-disila cyclohexane (MPSC) hydrochloride, a silicon-containing xenobiotic, in rat, dog, and man
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The metabolic pathways of Sandoz compound 58-112, 4-[(3-methoxyphenyl)methyl]-2,2,6,6-tetramethyl-1-oxa-4-aza-2,6-disila cyclohexane (MPSC) hydrochloride were evaluated in rat, dog, and man after a single oral dose. In rat, dog and man the major route of elimination was renal. In the dog, renal excretion of unchanged MPSC represented a substantial portion of the dose whereas in rat and man MPSC was completely metabolized prior to excretion. In rat and man, the major end-product metabolite was 3'-[{(hydroxydimethylsilyl)methylamino}methyl]-phenol glucuronide; 4-[(3-hydroxyphenyl)-methyl]-2,2,6,6-tetramethyl-1-oxa-4-aza-2,6-disil acyclohexane and 4-[(4-hydroxy-3-methoxyphenyl)methyl]-2,2,6,6-tetramethyl-1-oxa-4-aza- 2,6-disilacyclohexane and their conjugates were also present. In dog, the major end-product metabolites were the hippurate of 3-methoxybenzoic acid and 3-hydroxybenzoic acid.
- Dain,Nicoletti
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p. 951 - 961
(2007/10/03)
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- 29Si and 13C NMR Spectra of 4-Substituted 2-Methoxytrimethylsiloxybenzenes. Factors Determining the Chemical Shifts in Models of Lignin Constituents
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29Si and 13C NMR chemical shifts are reported for a series of twenty 4-substituted 2-methoxytrimethylsiloxybenzenes; the set of substituents incorporates a basic set of ten substituents differing in their relative polar and resonance effects and ten other
- Schraml, Jan,Kvicalova, Magdalena,Chvalovsky, Vaclav,Elder, Thomas,Brezny, Robert
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p. 973 - 978
(2007/10/02)
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- ASSIGNMENT OF 29Si NMR LINES AND DETERMINATION OF 29Si-13C COUPLING CONSTANTS IN PERTRIMETHYLSILYLATED LIGNIN-RELATED PHENOL-CARBOXYLIC ACIDS BY SELECTIVE HETERONUCLEAR INADEQUATE METHOD
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Selective heteronuclear (Si-C) INADEQUATE method has been successfully used to assign all 29Si chemical shifts in six pertrimethylsilylated lignin model compounds.Empirical assignment of the lines in the region δ=23-25 to trimethylsilyl esters of carboxyl
- Schraml, Jan,Past, Jaan,Puskar, Jueri,Pehk, Tonis,Lippmaa, Endel,Brezny, Robert
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p. 1985 - 1991
(2007/10/02)
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- Syntheses and Reactions of (Trimethylsiloxy)benzoyl Chlorides
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The (trimethylsiloxy)benzoyl chlorides 1-7 are easily to obtain under mild conditions from silylated hydroxybenzoic acids by means of thionyl chloride.Whereas these acid chlorides are stable at room temperature, they undergo condensation polymerisation at temperatures above 100 deg C.Reactions with various nucleophils, such as thioalcohols, phenols, amines, N,O-bis(silylated)amino acids and N-silylated lactams were investigated.With hexamethyldisilazane and thionyl chloride the (trimethylsiloxy)benzonitriles 21, 22 are accessible, and by means of trimethylsilylazide the (trimethylsiloxy)phenyl isocyanates 23-25 were obtained.Conversion with phenyl carbazate and subsequent silylation lead to the 2-(trimethylsiloxy)phenyl-1,3,4-oxadiazol-5-ones 27a-c.
- Schwarz, Gerd,Alberts, Heinrich,Kricheldorf, Hans R.
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p. 1257 - 1270
(2007/10/02)
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