Denis Tritsch

Isoprenoid biosynthesis via the methylerythritol phosphate pathway: the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB/IspH) from Escherichia coli is a [4Fe–4S] protein

The last enzyme (LytB) of the methylerythritol phosphate pathway for isoprenoid biosynthesis catalyzes the reduction of (E)‐4‐hydroxy‐3‐methylbut‐2‐enyl diphosphate into isopentenyl diphosphate and dimethylallyl diphosphate. This enzyme possesses a dioxygen‐sensitive [4Fe–4S] cluster. This prosthetic group was characterized in the Escherichia coli enzyme by UV/visible and electron paramagnetic resonance spectroscopy after reconstitution of the purified protein. Enzymatic activity required the presence of a reducing system such as flavodoxin/flavodoxin reductase/reduced nicotinamide adenine dinucleotide phosphate or the photoreduced deazaflavin radical.

Isoprenoid Biosynthesis through the Methylerythritol Phosphate Pathway: The (E)‐4‐Hydroxy‐3‐methylbut‐2‐enyl Diphosphate Synthase (GcpE) is a [4Fe–4S] Protein

nitrilotriaceticacidagarosecolumn. The enzyme was found to be 95% pure by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electro-phoresis) and presented an apparent molecular mass of43kDa. The purified protein was inactive, even in thepresenceofthereducingsystemsdetailedbelow.Suchalackofcatalyticactivitywasprobablyaresultofthepredominant

Isoprenoid biosynthesis as a target for antibacterial and antiparasitic drugs: phosphonohydroxamic acids as inhibitors of deoxyxylulose phosphate reducto-isomerase

Isoprenoid biosynthesis via the methylerythritol phosphate pathway is a target against pathogenic bacteria and the malaria parasite Plasmodium falciparum. 4-(Hydroxyamino)-4-oxobutylphosphonic acid and 4-[hydroxy(methyl)amino]-4-oxobutyl phosphonic acid, two novel inhibitors of DXR (1-deoxy-D-xylulose 5-phosphate reducto-isomerase), the second enzyme of the pathway, have been synthesized and compared with fosmidomycin, the best known inhibitor of this enzyme. The latter phosphonohydroxamic acid showed a high inhibitory activity towards DXR, much like fosmidomycin, as well as significant antibacterial activity against Escherichia coli in tests on Petri dishes.

Isoprenoid biosynthesis via the methylerythritol phosphate pathway. (E)-4-Hydroxy-3-methylbut-2-enyl diphosphate: chemical synthesis and formation from methylerythritol cyclodiphosphate by a cell-free system from Escherichia coli

Abstract 2- C -Methyl- d -erythritol cyclodiphosphate is converted into ( E )-4-hydroxy-3-methylbut-2-enyl diphosphate by a cell-free system from an Escherichia coli strain overexpressing the gcpE gene. The latter diphosphate, representing probably the last intermediate in the MEP pathway for isoprenoid biosynthesis, was identified by comparison with reference material obtained by chemical synthesis.

Chemical modification of horseradish peroxidase with ethanal-methoxypolyethylene glycol: Solubility in organic solvents, activity, and properties

Abstract The oxidation of polyethylene glycol monomethyl ethers (MW 350, 1990, and 5000) by the Moffatt-Swern method to the corresponding aldehyde is described. These aldehydes are used to modify horseradish peroxidase (HRP) by a reductive amination. The modification of two to three e-NH2 groups of the enzyme was observed. The isoelectric point of the native HRP (pI 8.8) was shifted to pI 5.5 on modification. The modified enzymes have an activity close to that of the native enzyme. Only the enzyme modified with the aldehyde MW 5000 (HRP 5000) was soluble and active in organic solvents like toluene, dioxane, and methylene chloride. In toluene, HRP 5000 was more sensitive to hydrogen peroxide inhibition than in buffer. At room temperature, it is more stable in toluene than in buffer.

Isoprenoid biosynthesis in Escherichia coli via the methylerythritol phosphate pathway: enzymatic conversion of methylerythritol cyclodiphosphate into a phosphorylated derivative of (E)-2-methylbut-2-ene-1,4-diol

Abstract A crude cell-free system from an Escherichia coli strain overexpressing the cluster containing the three genes yfgA, yfgB, and gcpE converted 2-C-methyl- d -erythritol 2,4-cyclodiphosphate ( 1 ) into a phosphorylated derivative of (E)-2-methylbut-2-ene-1,4-diol ( 6 ), which most probably represents a novel intermediate in the methylerythritol phosphate pathway for isoprenoid biosynthesis. The free diol 6 was accumulated by phosphatase treatment of the crude enzyme preparation and was identified by comparison with a synthetic reference.

The primary structure of ribosomal protein S7 from E. coli strains K and B.

Protein S7 from the small subunit of ~sc~ler~chi~ coli ribosomes has been reported to interact specifically with 16 S RNA (reviewed [ 11). The proportion of the RNA involved in the binding has been identified as part of the 3’-end [2-4]. On the other hand the part of the protein that binds to the RNA has been investigated by covalcntly linking S7 to the RNA. Protein 57 can be crosslinked to 16 S RNA by ultraviolet irradiation of the small subunit [5]. Ultraviolet irradiation experiments have also been made on an in vitro complex between protein S7 and 16 S RNA. The results indicated that at least four peptides were crosslinked to the RNA [6]. It can be expected that knowledge of the primary structure of protein S7 will contribute to a deeper understanding of the molecular mechanisms of protein-RNA interaction. It has been shown that E. coli strains differ in their S7 proteins [7-l 11. Ribosomes from strains B, C and MRE600 contain a protein S7 which differs extensively in size, charge, amino acid composition and im~~~unological properties from the protein S7 in strain K [ Ill. The amino acid sequences of protein S7 isolated from E. coli strain B and K are reported here. Protein S7B consists of 153 amino acids and protein S7K contains 177 amino acids. This difference of 24 amino acids between S7B and S7K occurs at the C-terminal end of the protein chain.

Plant isoprenoid biosynthesis via the MEP pathway: In vivo IPP/DMAPP ratio produced by (E)‐4‐hydroxy‐3‐methylbut‐2‐enyl diphosphate reductase in tobacco BY‐2 cell cultures

Feeding tobacco BY‐2 cells with [2‐13C,4‐2H]deoxyxylulose revealed from the 13C labeling that the plastid isoprenoids, synthesized via the MEP pathway, are essentially derived from the labeled precursor. The ca. 15% 2H retention observed in all isoprene units corresponds to the isopentenyl diphosphate (IPP)/dimethylallyl diphosphate (DMAPP) ratio (85:15) directly produced by the hydroxymethylbutenyl diphosphate reductase, the last enzyme of the MEP pathway. 2H retention characterizes the isoprene units derived from the DMAPP branch, whereas 2H loss represents the signature of the IPP branch. Taking into account the enantioselectivity of the reactions catalyzed by the (E)‐4‐hydroxy‐3‐methylbut‐2‐enyl diphosphate reductase, the IPP isomerase and the trans‐prenyl transferase, a single biogenetic scheme allows to interpret all labeling patterns observed in bacteria or plants upon incubation with 2H labeled deoxyxylulose.

Irreversible thermoinactivation of glucoamylase from Aspergillus niger and thermostabilization by chemical modification of carboxyl groups

The incubation of glucoamylase from Aspergillus niger at 70 degrees C induced its rapid and irreversible inactivation. The covalent modifications of the protein structure involved in the thermoinactivation depended on the pH of the medium. We observed the formation of a low amount of disulfide-linked oligomers showing that disulfide exchange takes place at pH 5.5. Hydrolysis of peptide bonds at pH 3.5 and 4.5 was also detected. The chemical modification of carboxyl groups with a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) decreased the rate of appearance of low-molecular-weight peptides at pH 3.5 and 4.5 upon heating at 70 degrees C. However, the rate of inactivation at such pH values was not modified. Modification of carboxyl groups with EDC in the presence of ethylenediamine leading to the transformation of three carboxyl groups to amino groups increased the thermostability of the enzyme for temperatures above the temperature of compensation, Tc, which is 60 degrees C.

Studies of the binding sites of Escherichia coli ribosomal protein S7 with 16S RNA by ultraviolet irradiation

It is now generally agreed that several ribosomal proteins, namely