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1.
ACS Catal ; 9(5): 3900-3905, 2019 May 03.
Article in English | MEDLINE | ID: mdl-31080689

ABSTRACT

Isomerization is a fundamental reaction in chemistry. However, isomerization of phenyl methyl ethers has not been described yet. Using a cobalamin-dependent methyl transferase, a reversible shuttle concept was investigated for isomerization of catechol monomethyl ethers. The methyl ether of substituted catechol derivatives was successfully transferred onto the adjacent hydroxy moiety. For instance, the cobalamin-dependent biocatalyst transformed isovanillin to its regioisomer vanillin with significant regioisomeric excess (68% vanillin). To the best of our knowledge, isomerization by methyl transfer employing a methyl transferase has not been reported before.

2.
Trends Biotechnol ; 33(7): 371-3, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25887333

ABSTRACT

Cobalamine cofactors (vitamin B12) are complex organometallic molecules that are crucial for the activity of a variety of different interesting enzymes such as isomerases, methyltransferases, and dehalogenases. Developments in understanding the structure, mechanism, and role in nature of methylcobalamin-dependent methyltransferases make them excellent candidates for biotechnological applications such as biocatalytic dealkylation.


Subject(s)
Biotechnology , Methyltransferases , Vitamin B 12 , Methylation
3.
Bioorg Med Chem ; 22(20): 5558-62, 2014 Oct 15.
Article in English | MEDLINE | ID: mdl-24951100

ABSTRACT

Valinol is part of numerous pharmaceuticals and has various other important applications. Optically pure valinol (ee >99%) was prepared employing different ω-transaminases from the corresponding prochiral hydroxy ketone. By the choice of the enzyme the (R)- as well as the (S)-enantiomer were accessible. Reductive amination was performed in organic solvent (MTBE) using 2-propyl amine as amine donor whereas alanine was applied in or in aqueous medium. Transformations in phosphate buffer were successfully performed even at 200 mM substrate concentration (20.4 g/L) leading to 99% (R) and 94% (S) conversion with perfect optical purity (>99% ee).


Subject(s)
Methyl Ethers/metabolism , Transaminases/metabolism , Valine/analogs & derivatives , Amination , Methyl Ethers/chemistry , Molecular Structure , Stereoisomerism , Transaminases/chemistry , Valine/biosynthesis , Valine/chemistry , Water/chemistry , Water/metabolism
4.
Chemistry ; 19(8): 2859-65, 2013 Feb 18.
Article in English | MEDLINE | ID: mdl-23292758

ABSTRACT

The regioselectivity of various enantiocomplementary ω-transaminases was evaluated for the stereoselective monoamination of designated 1,5-diketones; excellent conversions, enantio- and regioselectivities were observed. The resulting amino-ketones underwent spontaneous intramolecular ring closure to afford Δ1-piperideines, which served as precursors for the cis- and anti-piperidine scaffold as demonstrated for the synthesis of the alkaloids dihydropinidine and epi-dihydropinidine. Key to the success of accessing the trans-piperidines was a Lewis acid mediated conformational change of the Δ1-piperideines in the reduction step. Thus, all four diastereomers of 2,6-disubstituted piperidines could successfully be prepared.


Subject(s)
Alkaloids/chemistry , Ketones/chemistry , Lewis Acids/chemistry , Piperidines/chemistry , Piperidines/chemical synthesis , Transaminases/chemistry , Amination , Molecular Structure , Stereoisomerism
5.
European J Org Chem ; 2013(16): 3397-3402, 2013 Jun 01.
Article in English | MEDLINE | ID: mdl-25191103

ABSTRACT

A short and efficient total synthesis of the alkaloid isosolenopsin and its enantiomer has been achieved. In the key step, a ω-transaminase catalyzed the regioselective mono-amination of the diketone pentadecane-2,6-dione which was obtained in a single step via Grignard reaction. Initial low conversions in the biotransformation could be overcome by optimisation of the reaction conditions employing suitable cosolvents. In the presence of 20 vol% DMF or n-heptane best results were obtained employing two enantio-complementary ω-transaminases originating from Arthrobacter between 30-40 °C; under these conditions conversions of >99% and perfect stereocontrol (ee > 99%) were achieved. Diastereostelective chemical reduction (H2/Pd/C) of the biocatalytic product gave the target compound. The linear three step synthesis provided the natural product isosolenopsin in diastereomerically pure form (ee > 99%, d.r. = 99:1) with an overall yield of 64%.

7.
J Org Chem ; 70(23): 9168-74, 2005 Nov 11.
Article in English | MEDLINE | ID: mdl-16268586

ABSTRACT

[Structure: see text]. The IspG protein is known to catalyze the transformation of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate in the nonmevalonate pathway of isoprenoid biosynthesis. We have found that the apparent IspG activity in the cell extracts of recombinant Escherichia coli cells as observed by a radiochemical assay can be enhanced severalfold by coexpression of the isc operon which is involved in the assembly of iron-sulfur clusters. The recombinant protein was isolated by affinity chromatography under anaerobic conditions. With a mixture of flavodoxin, flavodoxin reductase, and NADPH as the reducing agent, stringent assay methods based on photometry or on 13C NMR detection of multiply 13C-labeled substrate/product ratios afforded catalytic activities greater than 60 nmol mg(-1) min(-1) for the protein "as isolated" (i.e., without reconstitution of any kind). Lower apparent activities were found using photoreduced deazaflavin as an artifactual electron donor, whereas dithionite was unable to serve as an artificial electron donor. The apparent Michaelis constant for 2-C-methyl-D-erythritol 2,4-cyclodiphosphate was 700 microM. The enzyme was inactivated by EDTA and could be reactivated by Mn2+. The pH optimum was at 9.0. The protein contained 2.4 iron ions and 4.4 sulfide ions per subunit. The replacement of any of the three conserved cysteine residues afforded mutant proteins which were devoid of catalytic activity and contained less than 6% of Fe2+ and less than 23% of S2- as compared to the wild-type protein. Sequence comparison indicates that putative IspG proteins of plants, the apicomplexan protozoan Plasmodium falciparum, and bacteria from the Bacteroidetes/Chlorobi group contain an insert of about 170-320 amino acid residues as compared with eubacterial enzymes.


Subject(s)
Escherichia coli Proteins , Terpenes/metabolism , Amino Acid Sequence , Catalysis , Chromatography, Affinity , Erythritol/analogs & derivatives , Erythritol/metabolism , Escherichia coli , Escherichia coli Proteins/genetics , Escherichia coli Proteins/isolation & purification , Escherichia coli Proteins/metabolism , Flavodoxin/metabolism , Iron/chemistry , Mevalonic Acid/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , NADH, NADPH Oxidoreductases/metabolism , NADP/metabolism , Nuclear Magnetic Resonance, Biomolecular , Recombinant Proteins/isolation & purification , Sequence Homology, Amino Acid , Sulfides/chemistry
8.
J Am Chem Soc ; 126(40): 12847-55, 2004 Oct 13.
Article in English | MEDLINE | ID: mdl-15469281

ABSTRACT

The ispH gene of Escherichia coli specifies an enzyme catalyzing the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl diphosphate into a mixture of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the nonmevalonate isoprenoid biosynthesis pathway. The implementation of a gene cassette directing the overexpression of the isc operon involved in the assembly of iron-sulfur clusters into an Escherichia coli strain engineered for ispH gene expression increased the catalytic activity of IspH protein anaerobically purified from this strain by a factor of at least 200. For maximum catalytic activity, flavodoxin and flavodoxin reductase were required in molar concentrations of 40 and 12 microM, respectively. EPR experiments as well as optical absorbance indicate the presence of a [3Fe-4S](+) cluster in IspH protein. Among 4 cysteines in total, the 36 kDa protein carries 3 absolutely conserved cysteine residues at the amino acid positions 12, 96, and 197. Replacement of any of the conserved cysteine residues reduced the catalytic activity by a factor of more than 70 000.


Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/metabolism , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Catalysis , Electron Spin Resonance Spectroscopy , Enzyme Activation , Escherichia coli/genetics , Escherichia coli Proteins/biosynthesis , Escherichia coli Proteins/genetics , Iron-Sulfur Proteins/biosynthesis , Iron-Sulfur Proteins/genetics , Mutagenesis, Site-Directed , Nuclear Magnetic Resonance, Biomolecular , Oxidoreductases/biosynthesis , Oxidoreductases/genetics , Plasmids/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrophotometry, Ultraviolet
9.
Eur J Biochem ; 271(13): 2658-69, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15206931

ABSTRACT

An open reading frame (Acc. no. P50740) on the Bacillus subtilis chromosome extending from bp 184,997-186,043 with similarity to the idi-2 gene of Streptomyces sp. CL190 specifying type II isopentenyl diphosphate isomerase was expressed in a recombinant Escherichia coli strain. The recombinant protein with a subunit mass of 39 kDa was purified to apparent homogeneity by column chromatography. The protein was shown to catalyse the conversion of dimethylallyl diphosphate into isopentenyl diphosphate and vice versa at rates of 0.23 and 0.63 micromol.mg(-1).min(-1), respectively, as diagnosed by 1H spectroscopy. FMN and divalent cations are required for catalytic activity; the highest rates were found with Ca2+. NADPH is required under aerobic but not under anaerobic assay conditions. The enzyme is related to a widespread family of (S)-alpha-hydroxyacid oxidizing enzymes including flavocytochrome b2 and L-lactate dehydrogenase and was shown to catalyse the formation of [2,3-13C2]lactate from [2,3-13C2]pyruvate, albeit at a low rate of 1 nmol.mg(-1).min(-1). Putative genes specifying type II isopentenyl diphosphate isomerases were found in the genomes of Archaea and of certain eubacteria but not in the genomes of fungi, animals and plants. The analysis of the occurrence of idi-1 and idi-2 genes in conjunction with the mevalonate and nonmevalonate pathway in 283 completed and unfinished prokaryotic genomes revealed 10 different classes. Type II isomerase is essential in some important human pathogens including Staphylococcus aureus and Enterococcus faecalis where it may represent a novel target for anti-infective therapy.


Subject(s)
Bacillus subtilis/enzymology , Isomerases/metabolism , Terpenes/metabolism , Amino Acid Sequence , Base Sequence , Catalysis , DNA Primers , Isomerases/chemistry , Isomerases/genetics , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Phylogeny , Sequence Homology, Amino Acid , X-Ray Diffraction
10.
Chem Biodivers ; 1(9): 1367-76, 2004 Sep.
Article in English | MEDLINE | ID: mdl-17191914

ABSTRACT

To investigate the unknown stereochemical course of the reaction catalyzed by the type-II isomerase, which interconverts isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), a sample of [1,2-(13)C2]-IPP stereospecifically labelled with 2H at C2 was prepared by incubating a D2O solution of (E)-4-hydroxy-3-methyl[1,2-(13)C2]but-2-enyl diphosphate with a recombinant IspH protein of Escherichia coli in the presence of NADH as a reducing agent and flavodoxin as well as flavodoxin reductase as auxiliary proteins. As monitored by 13C-NMR spectroscopy, treatment of the deuterated IPP with either type-I or type-II IPP isomerase resulted in the formation of DMAPP molecules retaining all the 2H label of the starting material. From the known stereochemical course of the type-I isomerase-catalyzed reaction, one has to conclude that the label introduced from D2O in the course of the IspH reaction resides specifically in the H(Si)-C2 position of IPP and that the two isomerases mobilize specifically the same H(Re)-C2 ligand of their common IPP substrate. The outcome of an additional experiment, in which unlabelled IPP was incubated in D2O with the type-II enzyme, demonstrates that the two isomerases also share the same preference in selecting for their reaction the (E)-methyl group of DMAPP.


Subject(s)
Hemiterpenes/analysis , Hemiterpenes/chemistry , Organophosphorus Compounds/analysis , Organophosphorus Compounds/chemistry , Bacillus subtilis/isolation & purification , Escherichia coli/isolation & purification , Escherichia coli Proteins/analysis , Escherichia coli Proteins/chemistry , Molecular Conformation
11.
Proc Natl Acad Sci U S A ; 100(4): 1586-91, 2003 Feb 18.
Article in English | MEDLINE | ID: mdl-12571359

ABSTRACT

Earlier in vivo studies have shown that the sequential action of the IspG and IspH proteins is essential for the reductive transformation of 2C-methyl-d-erythritol 2,4-cyclodiphosphate into dimethylallyl diphosphate and isopentenyl diphosphate via 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate. A recombinant fusion protein comprising maltose binding protein and IspG protein domains was purified from a recombinant Escherichia coli strain. The purified protein failed to transform 2C-methyl-d-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate, but catalytic activity could be restored by the addition of crude cell extract from an ispG-deficient E. coli mutant. This indicates that auxiliary proteins are required, probably as shuttles for redox equivalents. On activation by photoreduced 10-methyl-5-deaza-isoalloxazine, the recombinant protein catalyzed the formation of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate from 2C-methyl-d-erythritol 2,4-cyclodiphosphate at a rate of 1 nmol x min(-1) x mg(-1). Similarly, activation by photoreduced 10-methyl-5-deaza-isoalloxazine enabled purified IspH protein to catalyze the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate into a 6:1 mixture of isopentenyl diphosphate and dimethylallyl diphosphate at a rate of 0.4 micromol x min(-1) x mg(-1). IspH protein could also be activated by a mixture of flavodoxin, flavodoxin reductase, and NADPH at a rate of 3 nmol x min(-1) x mg(-1). The striking similarities of IspG and IspH protein are discussed, and plausible mechanistic schemes are proposed for the two reactions.


Subject(s)
Bacterial Proteins/metabolism , Escherichia coli Proteins , Oxidoreductases/metabolism , Polyisoprenyl Phosphates/biosynthesis , Xylulose/analogs & derivatives , Xylulose/metabolism , Blotting, Western , Catalysis , Chromatography, Affinity , Escherichia coli/metabolism , Nuclear Magnetic Resonance, Biomolecular
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