Structure and Substrate Specificity of S-Methyl Thiourocanate Hydratase.

Nicotinamide adenine dinucleotide (NAD+) is a common cofactor in enzyme-catalyzed reactions that involve hydride transfers. In contrast, urocanase and urocanase-like enzymes use NAD+ for covalent electrophilic catalysis. Deciphering avenues by which this unusual catalytic strategy has diversified by evolution may point to approaches for the design of novel enzymes. In this report, we describe the S-methyl thiourocanate hydratase (S-Me-TUC) from Variovorax sp. RA8 as a novel member of this small family of NAD+-dependent hydratases. This enzyme catalyzes the 1,4-addition of water to S-methyl thiourocanate as the second step in the catabolism of S-methyl ergothioneine. The crystal structure of this enzyme in complex with the cofactor and a product analogue identifies critical sequence motifs that explain the narrow and nonoverlapping substrate scopes of S-methyl thiourocanate-, urocanate-, thiourocanate-, and Nτ-methyl urocanate-specific hydratases. The discovery of a S-methyl ergothioneine catabolic pathway also suggests that S-methylation or alkylation may be a significant activity in the biology of ergothioneine.

Bacterial Degradation of Nτ-Methylhistidine.

The first three enzymatic steps by which organisms degrade histidine are universally conserved. A histidine ammonia-lyase (EC 4.3.1.3) catalyzes 1,2-elimination of the α-amino group from l-histidine; a urocanate hydratase (EC 4.2.1.49) converts urocanate to 4-imidazolone-5-propionate, and this intermediate is hydrolyzed to N-formimino-l-glutamate by an imidazolonepropionase (EC 3.5.2.7). Surprisingly, despite broad distribution in many species from all kingdoms of life, this pathway has rarely served as a template for the evolution of other metabolic processes. The only other known pathway with a similar logic is that of ergothioneine degradation. In this report, we describe a new addition to this exclusive collection. We show that the firmicute Bacillus terra and other soil-dwelling bacteria contain enzymes for the degradation of Nτ-methylhistidine to l-glutamate and N-methylformamide. Our results indicate that in some environments, Nτ-methylhistidine can accumulate to concentrations that make its efficient degradation a competitive skill. In addition, this process describes the first biogenic source of N-methylformamide.

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.

A variety of electrostatic interactions and adducts can activate NAD(P) cofactors for hydride transfer.

In NAD(P)-dependent enzymes the coenzyme gives or takes a hydride ion, but how the nicotinamide ring is activated to form the transition state for hydride transfer is not clear. On the basis of ultra-high resolution X-ray crystal structures of liver alcohol dehydrogenase (LADH) in complex with NADH and a number of substrate analogues we proposed that the activation of NADH is an integral part of the enzyme mechanism of aldehyde reduction [R. Meijers, R.J. Morris, H.W. Adolph, A. Merli, V.S. Lamzin, E.S. Cedergren-Zeppezauer, On the enzymatic activation of NADH, The Journal of Biological Chemistry 276(12) (2001) 9316-9321, %U http://www.ncbi.nlm.nih.gov/pubmed/11134046; R. Meijers, H.-W. Adolph, Z. Dauter, K.S. Wilson, V.S. Lamzin, E.S. Cedergren-Zeppezauer, Structural evidence for a ligand coordination switch in liver alcohol dehydrogenase, Biochemistry 46(18) (2007) 5446-5454, %U http://www.ncbi.nlm.nih.gov/pubmed/17429946]. We observed a nicotinamide with a severely distorted pyridine ring and a water molecule in close proximity to the ring. Quantum chemical calculations indicated that (de)protonation of the water molecule can be directly coupled to activation of NADH for hydride transfer. A systematic search of the Protein Data Bank (PDB) for atoms that come within van der Waals distance of the pyridine ring of the nicotinamide reveals that a large number of NAD(P)-containing protein complexes are involved in electrostatic interactions with the enzymatic environment. Using the deposited diffraction data to analyze the cofactor and its surroundings, we observe several adducts between protein atoms and the pyridine ring that were not previously reported. This further indicates that the enzymatic activation of NAD(P) induced by electrostatic interactions is an essential part of the hydride transfer mechanism.

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, 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.

Thermal activation of photoactivatable urocanase from Pseudomonas putida.

The dark inactivation of urocanase from Pseudomonas putida is caused by the formation of a sulfite adduct of the tightly bound coenzyme, nicotinamide adenine dinucleotide. Photodissociation of this adduct by UV radiation restores the enzyme activity. Based on cold exhaustive dialysis the modification reaction appeared to be irreversible. However, we now report that sulfite modification of urocanase is reversible at higher temperatures. An Arrhenius plot of the thermal activation is linear (20-38 degrees C). The activation energy for the enzyme activation is 114 kJ mol-1. The substance that is photodissociated from inactive urocanase reacts with urocanase to reform the modified enzyme indicating that sulfite is not oxidized, or otherwise changed through these processes. Nucleophiles (sulfite, hydroxylamine, hydride, cyanide) are known to inhibit urocanase by forming adducts with nicotinamide adenine dinucleotide. Urocanase inactivated by hydride or cyanide is not reactivated thermally or photochemically. Urocanase inactivated by hydroxylamine and by glycylglycine can be reactivated by a thermal reaction. In conclusion, sulfite-modified urocanase, which is formed in cells, can be reactivated not only by sunlight but also at physiological temperatures.

Proposal for the mechanism of action of urocanase. Inference from the inhibition by 2-methylurocanate.

Incubation of urocanase with 2-methylurocanate leads, after an initial normal reaction, to a time dependent inactivation of the enzyme. It is suggested that a tautomeric form of the product, 2-methyl-imidazolone propionate, is the actual inhibitor. On the basis of these and of published experimental data a novel mechanism is proposed for the urocanase reaction. The crucial and initial step is the electrophilic addition of enzyme-bound NAD to the 2-position of the imidazole nucleus of urocanate.

[Comparative effectiveness of the enteral and intravenous administration of rifampicin in treating experimental tuberculosis in rabbits]. / Sravnitel'naia éffektivnost' énteral'nogo i vnutrivennogo vvedeniia rifampitsina pri lechenii éksperimental'nogo tuberkuleza u krolikov.

The efficacy of intravenous and enteral administration of rifampicin was studied comparatively in treatment of hematogenic diseminated tuberculosis in rabbits. The drugs were administered in a dose of 10 mg/kg for 60 days. The functional state of the liver was studied in the time course by activity of urokinase, a hepatospecific enzyme and transaminases. Monotherapy with rifampicin capsules administered enterally resulted in death of 66.6 per cent of the animals due to tuberculosis during the first month of the treatment. Completion of the treatment course in 4 rabbits did not result in abacillation. When rifampicin was administered intravenously no deaths of the animals due to tuberculosis were recorded. Complete abacillation of the internal organs was observed in 37.5 per cent of the rabbits. The changes in activity of the indicator enzymes were transient and after discontinuation of the treatment came to normal.

Activation of urocanase from Pseudomonas putida by electronically excited triplet species.

Urocanase from Pseudomonas putida becomes inactive in growing and resting cells and, as shown previously, is activated by the direct absorption of ultraviolet light. In this study, we describe the activation of urocanase by energy transfer from triplet indole-3-aldehyde, generated in the peroxidase-catalyzed aerobic oxidation of indole-3-acetic acid. The activation was time-, temperature-, and pH-dependent. The involvement of reactive oxygen intermediates was excluded by the lack of effect of appropriate quenchers and traps. Triplet quenchers, in contrast, reduced the level of activation. Photoexcited rose bengal, a triplet species of a different nature and origin, was also effective in promoting activation. These results demonstrate a potential mechanism of urocanase regulation not dependent on an environmental source of light, but rather brought about by an enzymically generated excited species.

The effects of vitamin A deficiency on hepatic folate metabolism in rats.

The effects of severe vitamin A deficiency (liver retinol less than 2 micrograms/g) on hepatic folate metabolism in rats were studied. The oxidation of a [ring-2-14C] histidine load or a [14C]formate load to 14CO2 was significantly depressed in vitamin A-deficient rats and those given histidine also excreted more urinary formiminoglutamic acid (FiGlu) than pair-fed controls. The increase in FiGlu excretion was not due to augmented production from histidine, implicating an impairment of FiGlu catabolism. FiGlu formiminotransferase activity was unaltered in vitamin A-deficient rats, but hepatic tetrahydrofolic acid (THF) concentration was decreased by 58% in vitamin A-deficient rats given a histidine load while 5-methyl-THF concentration was increased by 39%. Formyl-THF and total folate levels were similar to controls. A redistribution of folate coenzymes was not found in vitamin A-deficient rats not force fed histidine. A 43% decrease in 10-formyl-THF dehydrogenase activity, which generates both THF and the 14CO2 from the labeled substrates, and an 81% increase in 5,10-methylene-THF reductase activity, which generates 5-methyl-THF, were found in vitamin A-deficient rats. It appears that the production of severe vitamin A deficiency results in selective changes in the activities of hepatic folate-dependent enzymes, so that when a load of a one-carbon donor is given, THF concentration decreases and metabolism of the load is impaired.