Gene cloning and characterization of thiourocanate hydratase from Burkholderia sp. HME13.

A novel enzyme, thiourocanate hydratase, which catalyses the conversion of thiourocanic acid to 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid, was isolated from the ergothioneine-utilizing strain, Burkholderia sp. HME13. When the HME13 cells were cultured in medium containing ergothioneine as the sole nitrogen source, thiourocanate-metabolizing activity was detected in the crude extract from the cells. However, activity was not detected in the crude extract from HME13 cells that were cultured in Luria-Bertani medium. The gene encoding thiourocanate hydratase was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. The enzyme showed maximum activity at pH 7.5 and 55°C and was stable between pH 5.0 and 10.5, and at temperatures up to 45°C. The Km and Vmax values of thiourocanate hydratase towards thiourocanic acid were 30 µM and 7.1 µmol/min/mg, respectively. The enzyme was strongly inhibited by CuCl2 and HgCl2. The amino acid sequence of the enzyme showed 46% identity to urocanase from Pseudomonas putida, but thiourocanate hydratase had no urocanase activity.

Moderate UV Exposure Enhances Learning and Memory by Promoting a Novel Glutamate Biosynthetic Pathway in the Brain.

Sunlight exposure is known to affect mood, learning, and cognition. However, the molecular and cellular mechanisms remain elusive. Here, we show that moderate UV exposure elevated blood urocanic acid (UCA), which then crossed the blood-brain barrier. Single-cell mass spectrometry and isotopic labeling revealed a novel intra-neuronal metabolic pathway converting UCA to glutamate (GLU) after UV exposure. This UV-triggered GLU synthesis promoted its packaging into synaptic vesicles and its release at glutamatergic terminals in the motor cortex and hippocampus. Related behaviors, like rotarod learning and object recognition memory, were enhanced after UV exposure. All UV-induced metabolic, electrophysiological, and behavioral effects could be reproduced by the intravenous injection of UCA and diminished by the application of inhibitor or short hairpin RNA (shRNA) against urocanase, an enzyme critical for the conversion of UCA to GLU. These findings reveal a new GLU biosynthetic pathway, which could contribute to some of the sunlight-induced neurobehavioral changes.

Mutations in the urocanase gene UROC1 are associated with urocanic aciduria.

Urocanase is an enzyme in the histidine pathway encoded by the UROC1 gene. This report describes the first putative mutations, p.L70P and p.R450C, in the coding region of the UROC1 gene in a girl with urocanic aciduria presenting with mental retardation and intermittent ataxia. Computed (in silico) predictions, protein expression studies and enzyme activity assays suggest that none of the mutations can produce a fully functional enzyme. The p.L70P substitution, which probably implies the disruption of an alpha-helix in the N-terminus, would alter its properties and therefore, its function. The p.R450C change would render impossible any interaction between urocanase and its substrate and would loss its enzyme activity. Consequently, these studies suggest that both mutations could alter the correct activity of urocanase, which would explain the clinical and biochemical findings described in this patient.

Designed protein-protein association.

The analysis of natural contact interfaces between protein subunits and between proteins has disclosed some general rules governing their association. We have applied these rules to produce a number of novel assemblies, demonstrating that a given protein can be engineered to form contacts at various points of its surface. Symmetry plays an important role because it defines the multiplicity of a designed contact and therefore the number of required mutations. Some of the proteins needed only a single side-chain alteration in order to associate to a higher-order complex. The mobility of the buried side chains has to be taken into account. Four assemblies have been structurally elucidated. Comparisons between the designed contacts and the results will provide useful guidelines for the development of future architectures.

Fasting-induced changes in ECL cell gene expression.

Gastric enterochromaffin-like (ECL) cells release histamine in response to food because of elevation of gastrin and neural release of pituitary adenylate cyclase-activating peptide (PACAP). Acid secretion is at a basal level in the absence of food but is rapidly stimulated with feeding. Rats fasted for 24 h showed a significant decrease of mucosal histamine despite steady-state expression of the histamine-synthesizing enzyme histidine decarboxylase (HDC). Comparative transcriptomal analysis using gene expression oligonucleotide microarrays of 95% pure ECL cells from fed and 24-h fasted rats, thereby eliminating mRNA contamination from other gastric mucosal cell types, identified significantly increased gene expression of the enzymes histidase and urocanase catabolizing the HDC substrate L-histidine but significantly decreased expression of the cellular L-histidine uptake transporter SN2 and of the vesicular monoamine transporter 2 (VMAT-2) responsible for histamine uptake into secretory vesicles. This was confirmed by reverse transcriptase-quantitative polymerase chain reaction of gastric fundic mucosal samples from fed and 24-h fasted rats. The decrease of VMAT-2 gene expression was also shown by a decrease in VMAT-2 protein content in protein extracts from fed and 24-h fasted rats compared with equal amounts of HDC protein and Na-K-ATPase alpha(1)-subunit protein content. These results indicate that rat gastric ECL cells regulate their histamine content during 24-h fasting not by a change in HDC gene or protein expression but by regulation of substrate concentration for HDC and a decreased histamine secretory pool.

Structure and action of urocanase.

Urocanase (EC 4.2.1.49) from Pseudomonas putida was crystallized after removing one of the seven free thiol groups. The crystal structure was solved by multiwavelength anomalous diffraction (MAD) using a seleno-methionine derivative and then refined at 1.14 A resolution. The enzyme is a symmetric homodimer of 2 x 557 amino acid residues with tightly bound NAD+ cofactors. Each subunit consists of a typical NAD-binding domain inserted into a larger core domain that forms the dimer interface. The core domain has a novel chain fold and accommodates the substrate urocanate in a surface depression. The NAD domain sits like a lid on the core domain depression and points with the nicotinamide group to the substrate. Substrate, nicotinamide and five water molecules are completely sequestered in a cavity. Most likely, one of these water molecules hydrates the substrate during catalysis. This cavity has to open for substrate passage, which probably means lifting the NAD domain. The observed atomic arrangement at the active center gives rise to a detailed proposal for the catalytic mechanism that is consistent with published chemical data. As expected, the variability of the residues involved is low, as derived from a family of 58 proteins annotated as urocanases in the data banks. However, one well-embedded member of this family showed a significant deviation at the active center indicating an incorrect annotation.

Cloning, sequencing, and expression of the cold-inducible hutU gene from the antarctic psychrotrophic bacterium Pseudomonas syringae.

A promoter-fusion study with a Tn 5-based promoter probe vector had earlier found that the hutU gene which encodes the enzyme urocanase for the histidine utilization pathway is upregulated at a lower temperature (4 degrees C) in the Antarctic psychrotrophic bacterium Pseudomonas syringae. To examine the characteristics of the urocanase gene and its promoter elements from the psychrotroph, the complete hutU and its upstream region from P. syringae were cloned, sequenced, and analyzed in the present study. Northern blot and primer extension analyses suggested that the hutU gene is inducible upon a downshift of temperature (22 to 4 degrees C) and that there is more than one transcription initiation site. One of the initiation sites was specific to the cells grown at 4 degrees C, which was different from the common initiation sites observed at both 4 and 22 degrees C. Although no typical promoter consensus sequences were observed in the flanking region of the transcription initiation sites, there was a characteristic CAAAA sequence at the -10 position of the promoters. Additionally, the location of the transcription and translation initiation sites suggested that the hutU mRNA contains a long 5'-untranslated region, a characteristic feature of many cold-inducible genes of mesophilic bacteria. A comparison of deduced amino acid sequences of urocanase from various bacteria, including the mesophilic and psychrotrophic Pseudomonas spp., suggests that there is a high degree of similarity between the enzymes. The enzyme sequence contains a signature motif (GXGX(2)GX(10)G) of the Rossmann fold for dinucleotide (NAD(+)) binding and two conserved cysteine residues in and around the active site. The psychrotrophic enzyme, however, has an extended N-terminal end.

Crc is involved in catabolite repression control of the bkd operons of Pseudomonas putida and Pseudomonas aeruginosa.

Crc (catabolite repression control) protein of Pseudomonas aeruginosa has shown to be involved in carbon regulation of several pathways. In this study, the role of Crc in catabolite repression control has been studied in Pseudomonas putida. The bkd operons of P. putida and P. aeruginosa encode the inducible multienzyme complex branched-chain keto acid dehydrogenase, which is regulated in both species by catabolite repression. We report here that this effect is mediated in both species by Crc. A 13-kb cloned DNA fragment containing the P. putida crc gene region was sequenced. Crc regulates the expression of branched-chain keto acid dehydrogenase, glucose-6-phosphate dehydrogenase, and amidase in both species but not urocanase, although the carbon sources responsible for catabolite repression in the two species differ. Transposon mutants affected in their expression of BkdR, the transcriptional activator of the bkd operon, were isolated and identified as crc and vacB (rnr) mutants. These mutants suggested that catabolite repression in pseudomonads might, in part, involve control of BkdR levels.

Cloning and sequencing of a 29 kb region of the Bacillus subtilis genome containing the hut and wapA loci.

Within the framework of an international project for the sequencing of the entire Bacillus subtilis genome, a 29 kb chromosome segment, which contains the hut operon (335 degrees) and the wapA gene, has been cloned and sequenced. This region (28,954 bp) contains 21 complete ORFs and one partial one. The 5th, 6th and 17th genes correspond to hutH encoding histidase, hutP encoding the positive regulator for the hut operon and wapA encoding a precursor of three major wall-associated proteins, respectively. A homology search for their products deduced from the 21 complete ORFs revealed that nine of them exhibit significant homology to known proteins such as urocanase (Pseudomonas putida), a protein involved in clavulanic acid biosynthesis (Streptomyces griseus), amino acid permeases (lysine, Escherichia coli; histidine, Saccharomyces cerevisiae; and others), beta-glucoside-specific phosphotransferases (E. coli and Erwinia chrysanthemi) and 6-phospho-beta-glucosidases (E. coli and Erw. chrysanthemi). Based on the features of the determined sequence and the results of the homology search, as well as on genetic data and sequence of the hut genes reported by other groups, it is predicted that the B. subtilis hut operon may consist of the following six genes (6th-1st), the last of which is followed by a typical rho-independent transcription terminator: hutP, hutH, EE57A (hutU) encoding urocanase, EE57B (hutI) encoding imidazolone-5-propionate hydrolase, EE57C (hutG) encoding formiminoglutamate hydrolase and EE57D (tentatively designated as hutM) possibly encoding histidine permease. Interestingly, the direction of transcription of these hut genes is opposite to that of the movement of the replication fork.

A revised map location for the histidine utilization genes in Pseudomonas putida.

The histidine utilization genes hutH and hutU of Pseudomonas putida ATCC 12633 have been mapped by interrupted mating and transduction to a location at approximately 43 minutes on the chromosome, closely linked to ser-800 and met-400 markers previously shown to be at 46 and 42 minutes, respectively. Since restriction enzyme mapping and cloning results have established that all genes associated with the hut pathway are contiguous, earlier maps of this strain which place these genes near 10 minutes on the chromosome in a superoperonic catabolic cluster are in error.

Cloning, expression and mutational analysis of the urocanase gene (hutU) from Pseudomonas putida.

The histidine-utilizing hutU gene was isolated from a lambda-EMBL3 phage of a genomic library from Pseudomonas putida nicII and subcloned into the expression vector pT7-7. Escherichia coli BL21 cells were transformed with the recombinant plasmid and produced a catalytically active protein, amounting to approximately 30% of the total protein in the crude cell-free extract. The addition of NAD+ to the growth medium ensured the full occupation of active sites by the cofactor. This requires a mechanism for the transport of NAD+ into E. coli cells. Using the overproducing mutant a new, fast and efficient isolation procedure is described which yields electrophoretically homogeneous urocanase within two days. The yield of pure enzyme, based on the culture volume, has been improved 50-80-fold compared with the traditional method. To investigate the possible role of cysteine residues in the catalysis or in the tight binding of the cofactor NAD+, six different mutants were prepared. In each mutant protein, one conserved cysteine was exchanged for alanine. The resulting clones were tested for the expression of urocanase with catalytic activity; the Km and Vmax values were determined. Only Cys410 was essential for catalysis. There was no detectable reconstitution or increase of activity after the addition of NAD+, either in the essential Cys/Ala mutant or the other mutant proteins. Electrospray-mass spectroscopy of the wild-type enzyme revealed that the coenzyme is not covalently bound to the protein and computational analysis showed no typical sequence for a mononucleotide-binding domain like the Rossman fold. To obtain urocanase apoenzyme, P. putida nicII was transformed with pGP1-2 and pTET7-U and grown in nicotinate-depleted medium. Like the mutant proteins, no activation of the apoform occurred after the addition of NAD+. These observations led us to postulate a new model for the non-covalent but tight binding of NAD+ to the enzyme by 'trapping' the cofactor while folding the nascent protein.

Isolation, sequencing and expression in E. coli of the urocanase gene from white clover (Trifolium repens).

The urocanase gene was detected in a clone obtained from a genomic library of white clover. The entire gene has been sequenced and expressed in the pT7-7/E. coli BL 21 (DE 3) system. The deduced sequence of the plant urocanase is 72% homologous with that of the well-characterized urocanase from Pseudomonas putida. The purification procedure, as well as kinetic and electrophoretic behaviour, of the new enzyme are described.

Analysis of the transcriptional activity of the hut promoter in Bacillus subtilis and identification of a cis-acting regulatory region associated with catabolite repression downstream from the site of transcription.

Levels of transcripts initiated at a hut promoter in Bacillus subtilis were analysed. The addition of histidine to the culture medium increased the level of the transcript sixfold. In the presence of histidine and glucose together, the level of the transcript was reduced to the level in the absence of induction. Furthermore, addition of a mixture of 16 amino acids to cultures of induced cells and of catabolite-repressed cells decreased levels of the transcript 16-fold and 2.6-fold, respectively. Thus, it appears that at least three regulatory mechanisms associated with induction, catabolite repression, and amino acid repression, control the transcriptional activity of the hut promoter. Expression of the hut promoter-lacZ fusions that contained various regions of the hutP gene and deletion analysis of the hutP region revealed a cis-acting sequence associated with catabolite repression that was located between positions +204 and +231 or around position +203.

Cloning and sequencing the urocanase gene (hutU) from Pseudomonas putida.

A clone harbouring the entire urocanase gene (hutU) was obtained from a genomic library of Pseudomonas putida using oligonucleotide probes synthesised on the basis of known flanking sequences. One subunit of urocanase consists of 556 amino acids and has a molecular mass of 60,771 Da.

Cloning and expression in Escherichia coli of histidine utilization genes from Pseudomonas putida.

A library of the Pseudomonas putida chromosome, prepared through the use of the cosmid pJB8 ligated to a partial Sau3A digest of bacterial DNA, followed by in vitro packaging into bacteriophage lambda particles, was used to construct a strain of Escherichia coli which contained the genes for histidine utilization. This isolate produced a repressor product and all five enzymes required in Pseudomonas spp. for histidine dissimilation, whereas none of these could be detected in the nontransduced parent E. coli strain. When this transductant was grown on various media containing histidine or urocanate as the inducer, it was observed that production of the cloned histidine degradative enzymes was influenced somewhat by the choice of nitrogen source used but not by the carbon source. The recombinant cosmid was isolated and found to consist of 21.1 kilobase pairs of DNA, with approximately 16 kilobase pairs derived from Pseudomonas DNA and the remainder being from the pJB8 vector. Digestion of this insert DNA with EcoRI provided a 6.1-kilobase-pair fragment which, upon ligation in pUC8 and transformation into an E. coli host, was found to encode histidine ammonia-lyase and urocanase. The inducible nature of this production indicated that the hut repressor gene also was present on this fragment. Insertional inactivation of the histidine ammonia-lyase and urocanase genes by the gamma-delta transposon has permitted location of these structural genes and has provided evidence that transcription proceeds from urocanase through histidine ammonia-lyase. Mapping of the 16-kilobase-pair Pseudomonas DNA segment with restriction enzymes and subcloning of additional portions, one of which contained the gene for formiminoglutamate hydrolase and another that could constitutively express activities for both imidazolone propionate hydrolase and formylglutamate hydrolase, has provided evidence for the organization of all hut genes.