Biochemical and genetic characterization of a gentisate 1, 2-dioxygenase from Sphingomonas sp. strain RW5.

A 4,103-bp long DNA fragment containing the structural gene of a gentisate 1,2-dioxygenase (EC 1.13.11.4), gtdA, from Sphingomonas sp. strain RW5 was cloned and sequenced. The gtdA gene encodes a 350-amino-acid polypeptide with a predicted size of 38.85 kDa. Comparison of the gtdA gene product with protein sequences in databases, including those of intradiol or extradiol ring-cleaving dioxygenases, revealed no significant homology except for a low similarity (27%) to the 1-hydroxy-2-naphthoate dioxygenase (phdI) of the phenanthrene degradation in Nocardioides sp. strain KP7 (T. Iwabuchi and S. Harayama, J. Bacteriol. 179:6488-6494, 1997). This gentisate 1,2-dioxygenase is thus a member of a new class of ring-cleaving dioxygenases. The gene was subcloned and hyperexpressed in E. coli. The resulting product was purified to homogeneity and partially characterized. Under denaturing conditions, the polypeptide exhibited an approximate size of 38.5 kDa and migrated on gel filtration as a species with a molecular mass of 177 kDa. The enzyme thus appears to be a homotetrameric protein. The purified enzyme stoichiometrically converted gentisate to maleylpyruvate, which was identified by gas chromatography-mass spectrometry analysis as its methyl ester. Values of affinity constants (Km) and specificity constants (Kcat/Km) of the enzyme were determined to be 15 microM and 511 s-1 M-1 x 10(4) for gentisate and 754 microM and 20 s-1 M-1 x 10(4) for 3, 6-dichlorogentisate. Three further open reading frames (ORFs) were found downstream of gtdA. The deduced amino acid sequence of ORF 2 showed homology to several isomerases and carboxylases, and those of ORFs 3 and 4 exhibited significant homology to enzymes of the glutathione isomerase superfamily and glutathione reductase superfamily, respectively.

Microbial reduction of aromatic carboxylic acids.

Several benzoic, cinnamic and phenylacetic acid derivatives were screened with 20 micro-organisms, mainly fungi, for the reduction of their carboxylic function. For all organisms several compounds were reduced in fairly good yields up to 80% to the corresponding alcohol. No general rule could be established, concerning the substitution pattern, as to which compounds were transformed to the alcohol. Generally the reactions were accomplished within 48-70 h. Only minor, if any, side products were detected. Dicarboxylic acids, such as phthalic or phenylglutaric acids and similar compounds could not be reduced by the microorganisms tested.

Investigation of the Alkaloid Pattern of Datura innoxia Plants by Capillary Gas-Liquid-Chromatography-Mass Spectrometry.

The alkaloid composition of DATURA INNOXIA plants was analysed by quartz capillary GLC and GLC-MS. Significant differences were found in the alkaloid pattern of the respective plant organs even between different aerial parts. More than 30 alkaloids could be identified. Besides previously known substances, a further 18 tropine ester alkaloids as well as hygrine and tyramine were identified. Seventeen of these alkaloids have not been formerly found in the genus DATURA. Additionally, two hitherto unknown hygrine derivatives, presumably isomeric N-methylpyrrolidinyl-hygrines, were characterized.

Preferential phosphorylation of high mobility group protein 17 in vitro by a nuclear protein kinase.

The ability of the high group proteins (HMG-1, 2, 14 and 17) to serve as substrate for protein kinases was investigated by incubating them with a cytoplasmic and nuclear kinase. In both cases phosphate was incorporated into all four HMG proteins. The amount of phosphate incorporated and the specificity for the four proteins was quite different for the two kinases. Whereas the cytoplasmic kinase phosphorylated the HMG-1 and 2 to a higher degree than HMG-14 and 17, the nuclear kinase exhibited a high specificity for the HMG-17, leaving the other three proteins with only a small amount. The high preference of a nuclear kinase for HMG-17 may be indicative of a specific phosphorylation occurring also in vivo.

Influence of histone phosphorylation upon histone-histone interactions studied in vitro.

Histones H2b and H3, phosphorylated in vitro with the catalytic subunit of protein kinase I from rabbit skeletal muscle, were used to estimate the influence of histone phosphorylation upon histone-histone complex formation. Stoichiometry and interaction affinity of the complexes H2a-H2b, H4-H2b, and H4-H3 were determined by using the continuous variation method based on circular dichorism or fluorescence intensity. All complexes exhibit a 1:1 stoichiometry in sodium phosphate or sodium chloride solution of pH 7.0. The association constants of the complexes containing phosphorylated H2b were only slightly reduced, whereas that with phosphorylated H3 was strongly reduced relative to those of the nonphosphorylated species.

Specificity of DNA basic polypeptide interactions. II+ Influence of aromatic amino acid residues investigated with agarose bound lysine copolypeptides.

Binding affinities towards DNA and base pair specificities of lysine copolymers, containing different amounts of Phe, Tyr or Trp residues, were estimated using a previously described chromatographic method. Incorporation of few aromatic residues into polylysine causes a decrease in the binding affinity, however, further raising the aromatic residue - lysine ratio results in a continous increase of affinity, which is most pronounced with the Tyr copolymers and not observed with polymers containing neutral aliphatic amino acid residues. AT-specificity increases concomitant with binding affinity in the case of the Tyr copolymers but not with the Phe copolymers. The interaction of DNA with the alternating Phe-Lys polymer is significantly stronger than with the random copolymer of equal residue composition. The molecular and conformational reasons determining specificity are discussed.

Resolution and purification of histones on homologous series of hydrocarbon-coated agaroses.

Hydrophobic chromatography on alkyl-agarose columns has been applied to the fractionation of histones. This paper describes: (a) a two-column method for the resolution of whole histone from calf thymus into its five main components (H1, H2a, H2b, H3 and H4), (b) a rapid one-step procedure for the isolation of the H3 fraction from whole histone, (c) an alternative one-step procedure for the resolution of H3 and H2a (which co-elute during gel exclusion chromatography on Biogel P-60). These experiments are also used for gaining further insight into the mechanism of action of hydrocarbon-coated agaroses.

Specifity of DNA-basic polypeptide interactions. Influence of neutral residues incorporated into polylysine and polyarginine.

An approach is described for evaluation of the specificity of basic polypeptides concerning the base pair composition of DNA. The polypeptides were covalently bound to CNBr activated agarose and two DNAs strongly different in base composition but of equal molecular weight were loaded and detached by a NaCl gradient. The difference in the NaCl concerntrations between the elution maxima of the two DNAs was taken as a measure for the recognition specificity. The results obtained confirmed the known AT- und GC-specificity of polylysine and polyarginine, respectively. Neutral residues incorporated into polylysine generally reduce the interaction affinity and also the AT-specificity of their host. This behavior is very pronounced with three homogeneous fractions of clupeine containing about one third of neutral aliphatic amino acids within clusters of arginine; the base pair specificity of these arginine copolymers was found to be practically nil.