A spectrophotometric assay of D-glucuronate based on Escherichia coli uronate isomerase and mannonate dehydrogenase.

Escherichia coli uronate isomerase and mannonate dehydrogenase were overexpressed in E. coli BL21(DE3)pLysS cells and purified to near-homogeneity. The kinetic properties of the two enzymes were investigated. The isomerase was found to be inhibited by EDTA and to be stimulated by Zn(2+), Co(2+), and Mn(2+), but not by Mg(2+) or Ca(2+). Both enzymes were used to develop a sensitive spectrophotometric assay, in which D-glucuronate is converted to D-mannonate with concomitant oxidation of NADH to NAD(+). The sensitivity of this assay permits the detection of less than 1 nmol D-glucuronate. This assay can also be used to determine the concentration of beta-glucuronides and glucuronate 1-phosphate after enzymatic hydrolysis of these compounds with beta-glucuronidase or alkaline phosphatase.

The regulatory region of the uxuAB operon in Escherichia coli K12.

The uxuAB operon is composed of two genes coding for enzymes involved in hexuronate degradation. This operon is negatively controlled by the uxuR and exuR regulatory gene products. Starting from uxu hybrid plasmids, a 300 bp Sau3A restriction fragment was isolated and shown to contain the entire uxu regulatory region using in vitro gene fusions that brought lac gene expression under the control of the transcriptional and translational signals of the uxuA gene. The nucleotide sequence of this fragment was established. The start of the uxu mRNA was localized by S1 mapping experiments (the presence of a CAP binding site upstream of the promoter was shown by DNAse I footprinting and in vitro titration). The N-terminal amino acid sequence of the purified uxuA-lacZ gene product allowed the mapping of the uxuA initiation codon at 115 bp from the start of transcription.

Genetic analysis of uxuR and exuR genes: evidence for ExuR and UxuR monomer repressors interactions.

The uxuAB operon is under the dual control of uxuR- and exuR-encoded repressors whereas the exu regulon genes are regulated by the sole ExuR repressor. Mutations affecting the two exuR and uxuR regulatory genes were selected to investigate the relationship between the two repressors. The isolation of exuR and uxuR negative dominant mutations on a multicopy plasmid indicated that the active form of the two repressors was multimeric. The introduction of a uxuR negative dominant allele into a wild-type strain resulted in a significant increase in exu gene expression. This unexpected effect may have been the consequence of the formation of hybrid repressor molecules. This protein must be composed of native ExuR+ subunits aggregated with altered UxuR subunits. The same interference was observed for the exuR negative dominant allele on uxu gene derepression. The hypothesis given here implies that the two regions of the ExuR and UxuR repressors involved in the subunit aggregation present enough homologies to allow the formation of hybrid repressor molecules.

Intraparticulate localization of some peroxisomal enzymes related to fatty acid beta-oxidation.

Male Wistar rats were given a diet containing 0.05% (w/w) LK-903 (alpha-methyl-p-myristyroxycinnamic acid 1-monoglyceride) for 2 weeks. The activities of four hepatic peroxisomal enzymes involved in the fatty acyl-CoA beta-oxidizing system were determined. The activities of fatty acyl-CoA oxidase, crotonase, beta-hydroxybutyryl-CoA dehydrogenase and thiolase were all increased about three times by administration of LK-903. The intraparticulate localizations of the four enzymes were then investigated by treatment of the purified peroxisomes with Triton X-100, by sonication, and by sucrose-density-gradient centrifugation after Triton X-100 treatment. The results suggest that thiolase is localized in the matrix of peroxisomes, that crotonase and beta-hydroxybutyryl-CoA dehydrogenase are located in the core, and that all or at least part of fatty acyl-CoA oxidase is associated with the core, though its association is weak.

Regulation of hexuronate system genes in Escherichia coli K-12: multiple regulation of the uxu operon by exuR and uxuR gene products.

New regulatory mutants of Escherichia coli K-1 carrying alterations of the uxuR gene were isolated and characterized. In the presence of superrepressed or derepressed uxuR mutations, mannonic hydrolyase (uxuA) and oxidoreductase relationship analyses suggested that the uxuR gene product acted as a repressor in the control of uxuA-uxuB operon expression. uxuR mutations were localized near min 97, and the following gene order was established: (argH)-uxuR-uxuB-uxuA-(thr). Properties of exuR (point and deletion) mutants showed that both exuR and uxuR regulatory gene products were involved in the control of the uxuA uxuB operon. Analysis of exuR uxuR double-derepressed mutants suggested that exuR and uxuR repressors act cooperatively to repress the uxu operon.

Regulation of Escherichia coli K-12 hexuronate system genes: exu regulon.

Two types of Escherichia coli K-12 regulatory mutants, partially or totally negative for the induction of the five catabolic enzymes (uronic isomerase, uxaC; altronate oxidized nicotinamide adenine dinucleotide: uxaB; mannonate hydrolyase, uxuA) and the transport system (exuT) of the hexuronate-inducible pathway, were isolated and analyzed enzymatically. Hexuronate-catabolizing revertants of the negative mutants showed a constitutive synthesis for some or all of these enzymes. Negative and constitutive mutations were localized in the same genetic locus, called exuR, and the following order for the markers situated between the min 65 and 68 was determined: argG--exuR--exuT--uxaC--uxaA--tolC. The enzymatic characterization of the pleiotropic negative and constitutive mutants of the exuR gene suggests that the exuR regulatory gene product exerts a specific and total control on the three exuT, uszB, and uxaC-uxaA operons of the galacturonate pathway and a partial control on the uxuA-uxuB operon of the glucuronate pathway. The analysis of diploid strains conatining both the wild type and a negative or constitutive allele of the exuR gene, as well as the analysis of thermosensitive mutants of the exuR gene, was in agreement with a negative regulatory mechanism for the control of the hexuronate system.

Construction and expression of hybrid plasmids containing Escherichia coli K-12 uxu genes.

The three genes of the Escherichia coli K-12 uxu region (uxu operon and regulatory gene) were isolated on a ColE1-uxu hybrid plasmid from the bank of Clarke and Carbon, and a restriction map of this region was established. In vitro recombination techniques were used to subclone the uxu restriction fragments into the plasmid vector pBR322 or pBR325. The various chimeric plasmids obtained were analyzed by restriction mapping and characterized genetically by introducing them in uxu mutant or wild-type strains. Differential rates of synthesis of the enzymes coded for by the uxu region were measured in the plasmid-containing strains; amplification of the products of the cloned genes was up to 40-fold the level found in haploid strains. The enzymes coded for by uxuA and uxuB were synthesized in vitro in a coupled transcription-translation system, confirming the results of the cloning experiments. The restriction analysis also suggests that the transcriptional direction of the uxu operon is from uxuA to uxuB and that the order of the loci in this region is: uxuR (regulatory gene), uxuB, uxuA, uxuAp (promoter), uxuAo (operator).