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Isolation and properties of prephenate dehydratase from Flavobacterium devorans r are described. The enzyme was enriched 43-fold by ammonium sulfate precipitation, by gel chromatography on ultrogel ACA-34 and by hydrophobic chromatography on decylagarose. The molecular weight was estimated to be 1.35 x 10(5). The enzyme was activated in 1mM solutions of L-tyrosine 4,4-fold, of L-phenylalanine 3.9fold and by L-tryptophan 1.9-fold in 0.02M Tris/HCl buffer at pH 7.7. The Km values were, when adding the activator, at L-tyrosine 1.5 x 10(-5) M, at L-phenylalanine 1.3 x 10(-5) M and at L-tryptophan 1 x 10(-5) M. Regulation of aromatic amino acid is discussed in connection with the prephenate dehydratase.

Two enzymes of chloramphenicol-resistant Flavobacterium strain CB 60 and strain CB 6 which catalyse the transamination of tyrosine, phenylalanine and tryptophan were enriched 43-fold and 31-fold. The molecular mass for the aromatic-amino-acid aminotransferases of both strains was found to be 120000 Da and the isoelectric point was at about pH 4.2-4.3. Both enzymes are not influenced by EDTA. A ping pong bi-bi-mechanism was obtained for the kinetic mechanism of the reaction. The aminotransferases of strain CB 60 and CB 6 differ in the pH optimum (pH 8.4-9.0 and 7.8-9.0), in the optimum of temperature (40.0 degrees C and 57.5 degrees C) and in a higher heat stability of the enzyme of strain CB 60 in comparison to that of strain CB 6. The catalytic activity of the enzyme of strain CB 60 is not influenced by cycloserine, contrary to the enzyme of CB 6 which is inhibited to 89%. Isonicotinohydrazide does not inhibit the enzyme of strain CB 6, but reduces the catalytic activity of the enzyme of strain CB 60 to 64%. Both enzymes are inhibited differently by phenylhydrazine [78% (CB 60) and 56% (CB 6)]. Data in percentages are related to 0.15 mM inhibitor. For the substrates tyrosine, phenylalanine, tryptophan and 2-oxoglutarate, the aminotransferase of strain CB 60 has Km values of 15.0, 16.6, 16.6 and 1.7mM, and for the enzyme of strain CB 6 Km values of 1.40, 1.33, 1.37 and 0.97mM were obtained.

The tyrosine aminotransferase of Flavobacterium strain CB 60, strain CB 6 and F. devorans r - a partially purified enzyme was used - is able to deaminate oxidatively p-aminophenylalanine and the intermediate products of chloramphenicol degradation p-nitrophenylserine and p-aminophenylserine. The aminotransferases of the strains CB 6 and CB 60 also convert p-aminophenylserinol. p-Nitrophenylserinol only reacts with the enzyme from strain CB 6. Determination of substrate specificity from strain CB 6 shows that an alcoholic group in C3 position (ring proximal) and to a lower degree an alcoholic group in C1 position (ring distal) decrease the turnover rate. Based on its broad substrate specificity the tyrosine aminotransferase has the ability not only to metabolize physiological compounds but also degradation products of chloramphenicol.

Growth of a chloramphenicol-resistant mutant of Flavobacterium devorans (F. devorans r) was inhibited by L-phenylalanine and L-tyrosine and furthermore by L-tryptophan, 4-amino-benzoate, anthranilate and homogentisate. The inhibitionwas reversed partially by simultaneous addition of aromatic amino acids and 4-amino-benzoate. After cultivating F. devorans r in a glucose-minimal medium containing L-phenylalanine, the metabolites L-tyrosine, 4-hydroxyphenylpyruvate, 4-hydroxyphenylacetate, 4-hydroxybenzaldehyd, 4-hydroxybenzoate, homogentisate and anthranilate were found. Growth inhibition and a hypothetical degrading pathway of phenylalanine are discussed.

From the highly chloramphenicol-resistant cytophaga-like bacterium Flavobacterium CB60, which can both acetylate chloramphenicol and degrade it in co-metabolism, the chloramphenicol acetyltransferase (CAT) was purified to homogeneity and characterized. The purification included fractional precipitation with ammonium sulphate and two affinity chromatography steps, eluting CAT the first time with 5 mM-chloramphenicol and the second time with a linear gradient (0-10 mM) of chloramphenicol. The purification was 3979-fold. Properties of this CAT were investigated and compared with CATs from other bacteria. Although CAT from Flavobacterium CB60 shares some properties with the enzymes from Escherichia coli and other Gram-negative bacteria--especially with CATII and CATIII--it has distinct properties like extreme heat lability and the inability to produce diacetylchloramphenicol, so that it might be regarded as a new variant.

The chloramphenicol resistance of some flavobacteria was investigated comparatively. This resistance can be explained either by acetylation of chloramphenicol to O-acetylchloramphenicol via constitutively formed acetyltransferases, followed by cometabolic degradation (strain CB 60), or by limited uptake and total degradation (strain CB 6) by inducible enzymes or by other mechanisms (F. devorans). The mechanisms of resistance, CM-acetylation, CM-degradation and limited uptake are discussed.