Shifting the optimal pH of activity for a laccase from the fungus Trametes versicolor by structure-based mutagenesis.

Laccases are oxidizing enzymes of interest because of their potential environmental and industrial applications. We performed site-directed mutagenesis of a laccase produced by Trametes versicolor in order to improve its catalytic properties. Considering a strong interaction of the Asp residue in position 206 with the substrate xylidine, we replaced it with Glu, Ala or Asn, expressed the mutant enzymes in the yeast Yarrowia lipolytica and assayed the transformation of phenolic and non-phenolic substrates. The transformation rates remain within the same range whatever the mutation of the laccase and the type of substrate: at most a 3-fold factor increase was obtained for k(cat) between the wild-type and the most efficient mutant Asp206Ala with 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic) acid as a substrate. Nevertheless, the Asn mutation led to a significant shift of the pH (DeltapH = 1.4) for optimal activity against 2,6-dimethoxyphenol. This study also provides a new insight into the binding of the reducing substrate into the active T1 site and induced modifications in catalytic properties of the enzyme.

Cleavage of the diketonitrile derivative of the herbicide isoxaflutole by extracellular fungal oxidases.

Isoxaflutole is a herbicide activated in soils and plants to its diketonitrile derivative, the active herbicide principle. The diketonitrile derivative undergoes cleavage to the inactive benzoic acid analogue. In this paper, it is established that an oxidative mechanism implicating two successive reactions in the presence of dimethyldioxirane can chemically initiate the cleavage of the diketonitrile. It is also shown that two white rot strains, Phanerochaete chrysosporium and Trametes versicolor, are able to convert the diketonitrile to the acid when cultured in liquid media. This main metabolite amounts to 24.6 and 15.1% of initial herbicide content after 12-15 days of culture. Another polar metabolite represents <3.7% of the parent compound amount during the same period. Oxidative enzymes produced by the fungi show a time course similar to that of diketonitrile degradation. Purified laccase (EC 1. 10.3.2), in the presence of 2 mM 2, 2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) acting as a redox mediator at pH 3 supports the reaction with rates of 0.3-0.4 nmol h(-)(1) unit(-)(1).

Mass spectrometric determination of the cleavage sites in Escherichia coli dihydroorotase induced by a cysteine-specific reagent.

Escherichia coli dihydroorotase contains six cysteines/subunit, which are potential ligands of structural and catalytic zinc metals at protein sites of the enzyme. Specific thiol reagents modify, in nondenaturing conditions only, two of these cysteines; these two residues are thought to be ligands of structural zinc. We report here on the localization of these two cysteines on the polypeptide chain through their cyanylation by 2-nitro-5-thiocyanobenzoic acid (NTCB) and the analysis by mass spectrometry of the protein adducts. This is the first study of E. coli dihydroorotase by mass spectrometry, allowing the accurate determination of the subunit molecular weight (38,695). Treatment of dihydroorotase by NTCB induced a cleavage N-terminal to the cyanylated cysteines. The resulting fragments visualized on electrophoresis gel have been N-terminal sequenced, and their masses were determined by electrospray-ionizing mass spectrometry. This allowed the identification of cysteines 221 and 265 as the two residues cyanylated by the reagent NTCB. Results from gel filtration of dihydroorotase cyanylated on the two cysteines indicate that these residues are involved in subunit interactions leading to the active dimer. Consistent with literature data, we assume that cysteine 221 and cysteine 265, along with the neighboring cysteines 263 and 268 arranged in cluster, are potential ligands of structural zinc of E. coli dihydroorotase.

Assay of Escherichia coli dihydroorotase with enantiomeric substrate: practical preparation of carbamyl L-aspartate and high-performance liquid chromatography analysis of catalysis product.

A reasonably facile and effective procedure is described for preparing the optically active substrate of dihydroorotase (EC 3.5.2.3), N-carbamyl-L-aspartate (L-CA), which is not commercially available. Compared to the previously described methods, this procedure is not plagued by side reactions, and the L-CA is obtained in a solid form as the Mg2+/K+ mixed salt. In that salt form, the L-CA can be prepared in large quantity, and it is easier to handle and stable upon storage. L-CA can be separated from aspartate and its cyclic derivatives, dihydroorotate and hydantoin, by HPLC using a strong anion-exchange column. This HPLC method, which is used to check the purity of the synthesized carbamyl aspartate, is also effective for monitoring the enzymatic reaction.

Have deoxystreptamine aminoglycoside antibiotics the same binding site on bacterial ribosomes?

(3H) Tobramycin was used as a probe to determine the relationship between the structure of aminoglycoside antibiotics and their ability to remove this drug from its higher affinity binding site on the ribosome. The dissacharide moieties (neamine, tobramine, gentamine) appeared to have a common binding site, whereas the kanosamine, garosamine and ribose moieties determined the specificity of this binding. Amikacin and butikacin behaved in an anomalous manner in spite of their close structural relationship to tobramycin.