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PubMed1980

Multiple forms of boar, bull, and human acrosin.

Analytical disk gel electrophoresis with staining techniques for amidohydrolase activity at pH 7.6 demonstrated that partially purified acrosomal extracts of ejaculated bull, boar, and human spermatozoa contained three, apparently four, and two fractions, respectively, with acrosin-like activity. Acrosin amidohydrolase activity is present in the gels incubated in the staining medium at pH 5.0. Some methods for the extraction of human acrosin have been compared. These consist essentially of the extraction by detergent treatment and the extraction by acid procedures. Acid extraction of human spermatozoa yields a higher amount of acrosin than does detergent extraction; the acrosin specific activity, extracted by these methods, seems to be similar.

Acrosin↗

PubMed2000

Carrier-mediated uptake of the endogenous cannabinoid anandamide in RBL-2H3 cells.

Anandamide (N-arachidonylethanolamide) is an endogenous cannabinoid that mimics the pharmacologic effects of Delta(9)-tetrahydrocannabinol, the major bioactive substance in marijuana. Anandamide appears to be synthesized, released, and inactivated by mechanisms similar to those for other neurotransmitters. Of interest to the present studies are reports that anandamide undergoes carrier-mediated uptake into neuronal or glial cells after release, followed by rapid intracellular degradation by the intracellular fatty acid amidohydrolase. In addition to effects in the brain, anandamide has multiple effects in the periphery, particularly on cells of the immune system that express both a peripheral cannabinoid receptor and amidohydrolase enzyme. We have performed a detailed characterization of anandamide uptake in the cognate mast cell line RBL-2H3 to test the hypothesis that the uptake system in peripheral cells is also carrier-mediated and functionally similar to that observed in the central nervous system. RBL-2H3 cells exhibited robust, saturable transport of [(3)H]anandamide that was both time- and temperature-sensitive. This transport activity was not dependent on extracellular ion gradients for uptake and was inhibited selectively by other fatty acid-derived molecules, anandamide congeners, and the psychoactive cannabinoids such as Delta(9)-tetrahydrocannabinol. We conclude that anandamide transport in the RBL-2H3 cells is carrier-mediated, and uptake in peripheral cells is functionally and pharmacologically identical with that observed in neurons and astrocytes.

Amidohydrolases↗

PubMed1983

A new simple enzymatic assay method for urinary polyamines in humans.

We developed a new simple enzymatic assay method for measuring urinary polyamines (total amount of putrescine, spermidine, and cadaverine), using an acylpolyamine amidohydrolase and a putrescine oxidase. First conjugated polyamines (putrescine, spermidine, and cadaverine) in urine were hydrolyzed by incubation with an acylpolyamine amidohydrolase at 30 degrees for 1 hr. then, free polyamines were separated by cation-exchange chromatography and incubated with a putrescine oxidase at 30 degrees for 30 min. Hydrogen peroxide formed in this reaction was measured spectrophotometrically (at 514 nm). Polyamine levels in urine were determined in 70 normal subjects, 124 patients with cancer, and 52 patients with diseases other than cancer. Elevation above 3 S.D.s of the normal mean was found in 90 (72.6%) of the 124 patients with cancer and in 6 (11.5%) of the 52 patients with diseases other than cancer. Serial studies in 19 patients with cancer indicated that polyamines in urine were reduced after successful surgery. Our new method is simple and rapid and therefore very useful for routine clinical application. Moreover, our data indicate that the determination of polyamine levels is useful as a marker of disease activity in patients with cancer.

Amidohydrolases↗

PubMed1981

Combined enzymic-Jaffé method for determination of creatinine in serum.

Concentrations of creatinine, as determined in serum by a method involving the combined use of creatinine amidohydrolase (EC 3.5.2.10) and alkaline sodium picrate were found to be factitiously low, owing to a reversal of the enzyme reaction. This effect could be eliminated by converting creatine, the product of the enzymic reaction, to creatine phosphate. The combined enzymic-Jaffé method was therefore modified to include creatine amidohydrolase, creatine kinase, ATP, and Mg2+ in the reaction mixture. The modified method has good precision. We saw no significant interferences by relatively high concentrations of acetone, acetylacetone, ADP, creatine, creatine phosphate, glucose, glycocyamine, or pyruvate. Likewise, no interferences were evident with icteric, lipemic, or hemolytic serum samples. There was an excellent agreement between creatinine values obtained with our method and by a reference method based on isotope dilution-mass fragmentography. Our method is considerably simpler than the fully enzymic method for determination of creatinine and might be a method of choice if a high accuracy is desired.

Adenosine Triphosphate↗

PubMed1976

Role of enzymatically catalyzed 5-iodo-5,6-dihydrouracil ring hydrolysis on the dehalogenation of 5-iodouracil.

Incubation of 5-iodo-5,6-dihydrouracil (IH2Ura) with soluble rat liver enzymes at 37 degrees, pH 8.2, results in the rapid release of iodide ion. The second product resulting from the carbon skeleton of the dihydropyrimidine ring system is 2-amino-2-oxazoline-5-carboxylic acid (I). Ultraviolet absorbance measurements at 225 nm, where both IH2Ura and iodide ion absorb, indicate that IH2Ura dehalogenation is a two-step process. The first step, which is enzyme-dependent, involves dihydropyrimidine amidohydrolase (EC 3.5.2.2.)-catalyzed hydrolysis of the IH2Ura ring system presumably to yield 2-iodo-3-ureidopropionate. The enzyme preparations also catalyze the hydrolysis of 5-bromo-5,6-dihydrouracil, 5,6-dihydrouracil, and 5,6-dihydrothymine, the latter two of which are the natural substrates for dihydropyrimidine amidohydrolase. The second step in IH2Ura dehalogenation involves the nonenzymatically catalyzed, pH-independent intramolecular cyclization of 2-iodo-3-ureidopropionate via nucleophilic attack of the ureido oxygen atom on carbon-2 resulting in iodide ion and the oxazoline (I) as final products. The results are discussed relative to the role of pyrimidine catabolizing enzymes in 5-halopyrimidine dehalogenation.

Amidohydrolases↗

PubMed1999

Stereoselective metabolism of dexrazoxane (ICRF-187) and levrazoxane (ICRF-186).

A chiral HPLC method has been developed to separate razoxane (ICRF-159) in blood plasma into its enantiomers dexrazoxane (ICRF-187) and levrazoxane (ICRF-186). Dexrazoxane is clinically used as a doxorubicin cardioprotective agent and little is known of its in vivo metabolism. After intravenous administration of 20 mg/kg of razoxane to rats, the razoxane was eliminated from the plasma with a half-time of approximately 20 min. The levrazoxane:dexrazoxane ratio continuously increased with time to a value of 1.5 at 150 min, indicating that dexrazoxane is metabolized faster than levrazoxane. These results, confirmed with studies on liver supernatants, are consistent with the hypothesis that dihydropyrimidine amidohydrolase in the liver and kidney is responsible for the preferential metabolism of dexrazoxane in the rat compared to levrazoxane. It is possible that on a dose-per-dose basis marginally higher therapeutic levels of levrazoxane might be achieved in the heart tissue for a longer time compared to dexrazoxane due to dihydropyrimidine amidohydrolase-based metabolism in the liver and kidney. However, given the relatively small difference in elimination of the two enantiomers, it would be difficult to predict from this study whether or not dexrazoxane or levrazoxane might be more efficacious in reducing cardiotoxicity.

Animals↗

PubMed1994

Crystallization and preliminary X-ray analysis of the two domains of glucosamine-6-phosphate synthase from Escherichia coli.

The glutamine amidohydrolase and fructose 6-phosphate binding domains of glucosamine-6-phosphate synthase from Escherichia coli have been overexpressed, purified and crystallized for X-ray diffraction analysis. The crystals of the glutamine amidohydrolase domain belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 70.4 A, b = 82.5 A, c = 86.1 A, with two molecules in the asymmetric unit, and diffract to 1.9 A resolution. The native Patterson indicated pseudo c-face centering of the unit cell. The fructose 6-phosphate binding domain was crystallized in the hexagonal space group P6(1) or P6(5) with cell dimensions a = b = 63.5 A, c = 334.3 A and with two molecules in the asymmetric unit. Diffraction data to 2.6 A resolution have been collected.

Binding Sites↗

PubMed2006

N-acyl homoserine lactones are degraded via an amidolytic activity in Comamonas sp. strain D1.

Comamonas strain D1 enzymatically inactivates quorum-sensing (QS) signal molecules of the N-acyl homoserine lactone (N-AHSL) family, and exhibits the broadest inactivation range of known bacteria. It degrades N-AHSL with acyl-side chains ranging from 4 to 16 carbons, with or without 3-oxo or 3-hydroxy substitutions. N-AHSL degradation yields HSL but not N-acyl homoserine: strain D1 therefore harbors an amidohydrolase activity. Strain D1 is the fifth bacterium species in which an N-AHSL amidohydrolase is described. Consistent with its N-AHSL degradation ability, strain D1 efficiently quenches various QS-dependent functions in other bacteria, such as violacein production by Chromobacterium violaceum and pathogenicity and antibiotic production in Pectobacterium.

4-Butyrolactone↗

PubMed1996

Enzymes for anandamide biosynthesis and metabolism.

Anandamide is an endogenous ligand for cannabinoid receptors. We tried to isolate and purify "anandamide amidohydrolase' which hydrolyzes anandamide to arachidonic acid and ethanolamine. The enzyme activity was found in the microsomal fraction of porcine brain homogenate. The enzyme was solubilized in 1% Triton X-100, and partially purified by hydrophobic chromatography to a specific activity of about 0.3 mumol/min per mg protein (37 degrees C). Apparent K(m) for anandamide was about 60 microM. The enzyme reacted also with ethanolamides of linoleic, oleic, and palmitic acids at lower rates. This enzyme preparation also converted arachidonic acid to anandamide in the presence of 250 mM concentration of ethanolamine. Several lines of evidence including experiments using various inhibitors suggested that the anandamide synthase and amidohydrolase activities were derived from a single enzyme protein.

Animals↗

PubMed2005

Construction, purification, and functional characterization of His-tagged Candida albicans glucosamine-6-phosphate synthase expressed in Escherichia coli.

Expression plasmids containing recombinant genes encoding three His(6)-tagged versions of the enzyme, glucosamine-6-phosphate synthase from Candida albicans, were constructed and overexpressed in Escherichia coli. The gene products were purified by metal-affinity chromatography to near homogeneity with 77-80% yield and characterized in terms of size and enzymatic properties. Presence of oligohistidyl tags at either of two ends did not affect enzyme quarternary structure but strongly influenced its catalytic activity. The His6-N-tagged enzyme completely lost an ability of glucosamine-6-phosphate formation and amidohydrolase activity but retained the hexosephosphate-isomerising activity. On the other hand, two His6-C-tagged versions of glucosamine-6-phosphate synthase exhibited amidohydrolase activity almost equal to that of the wild-type enzyme but only 18% of its hexosephosphate-isomerising activity and about 1.5% of the synthetic activity.

Candida albicans↗

PubMed2002

Formation and structure of 1:1 complexes between aryl hydroxamic acids and vanadate at neutral pH.

Although aryl hydroxamic acids are well-known to form coordination complexes with vanadate (V(V)), the nature of these complexes at neutral pH and submillimolar concentrations, the conditions under which such complexes inhibit various serine amidohydrolases, is not well established. A series of qualitative and quantitative experiments, involving UV/vis, (1)H NMR, and (51)V NMR spectroscopies, established that both 1:1 and 1:2 vanadate/hydroxamate complexes form at pH 7.5, with the former dominating at submillimolar concentrations. Formation constants for the complexes of several aryl and alkyl hydroxamic acids were determined; for example, for benzohydroxamic acid, the stepwise formation constants of the 1:1 and 1:2 complexes were 3000 and 400 M(-1), respectively. The (51)V chemical shift of the 1:1 4-nitrobenzohydroxamic acid complex was -497 ppm, and that of its unsubstituted analogue was -498 ppm. A (1)H-(15)N HSQC spectrum of the 4-nitrobenzo-(15)N-hydroxamic acid/vanadate complex indicated the presence of an N-H group with (15)N and (1)H chemical shifts of 115 and 5.83 ppm, respectively. A (13)C NMR spectrum of the complex of 4-nitrobenzo-(13)C-hydroxamic acid with vanadate displayed a resonance at 170.1 ppm and thus a coordination-induced shift (CIS) of +3.8 ppm. In contrast, the CIS value of an established 1:2 complex, thought to contain chelated hydroxamic acid ligands, was +11.9 ppm. These spectral data led to the following structural picture of 1:1 complexes of vanadate and aryl hydroxamic acids. They contain penta- or hexa-coordinated vanadium. The ligand is in the hydroxamate rather than hydroximate form. The ligand is presumably bound to vanadium through the hydroxamic hydroxyl oxygen, but the hydroxamic acid carbonyl oxygen interacts weakly with vanadium. These species are the most likely candidates for the inhibitors of serine amidohydrolases found in vanadate/hydroxamic acid mixtures.

Hydrogen-Ion Concentration↗

PubMed1998

Exploration of biologically relevant conformations of anandamide, 2-arachidonylglycerol, and their analogues using conformational memories.

The endogenous cannabinoid anandamide (N-arachidonoylethanolamide) has been shown to possess higher affinity for the cannabinoid CB1 receptor than for the CB2 receptor. Carrier-mediated transport has been proposed to be essential for the termination of the biological effects of anandamide, while hydrolysis of anandamide is performed by a membrane-bound amidohydrolase, fatty acid amidohydrolase (FAAH). As interaction of anandamide with each of these targets occurs in different environments, the conformations of anandamide for interaction with each target may differ. To ascertain what conformations of anandamide, a highly flexible molecule, are favored in polar and nonpolar environments, the new method of Conformational Memories (CM) was used. CM has been shown to achieve complete conformational sampling of highly flexible ligands, to converge in a very practical number of steps, and to be capable of overcoming energy barriers very efficiently (Guarnieri et al. J. Am. Chem. Soc. 1996, 118, 5580). The generalized Born/surface area (GB/SA) continuum solvation models for chloroform and for water were used in the CM calculations. As a means of validation, CM was first applied to arachidonic acid because both experimental and theoretical conformational studies of arachidonic acid have been reported. CM was also applied to sn-2-arachidonylglycerol (2-AG), another endogenous CB ligand; to a 1,1-dimethylheptyl derivative of anandamide, an analogue with higher CB1 affinity than anandamide; and to N-(2-hydroxyethyl)prostaglandin-B2-ethanolamide (PGB2-EA), a prostanoid ligand which does not bind to CB1. Consistent with the literature, arachidonic acid was found to exist in an extended, angle-iron shape and in back-folded conformations which were U, J, or helical in shape. The angle-iron and U-shapes were both highly populated conformations with the angle-iron preferred in CHCl3 and the U-shape preferred in H2O. Results for anandamide and 2-AG paralleled those for arachidonic acid with the exception that anandamide in water does not adopt a pure extended conformation but, rather, favors a hybrid-extended/U-shape. For the dimethyl-heptyl derivative of anandamide, the U-shape was found to be predominant in both environments (42% in CHCl3, 38% in H2O), but the population of the angle-iron shape was still significant (25% in CHCl3, 29% in H2O). For all of these ligands, J-shaped conformers constituted from 7% to 17% of the conformer population, while the helical shape was less than 5%. In both CHCl3 and H2O, the presence of the five-membered ring attenuates the ability of PGB2-EA to adopt an extended conformation. PGB2-EA was found instead to exist predominantly in an L-shape (i.e., distorted U-shape). The low probability of PGB2-EA adopting an extended conformation may be why PGB2-EA shows such low affinity for the CB1 receptor. The conformational information obtained here for anandamide and 2-AG may be useful in the design of rigid analogues which mimic the preferred molecular conformations (shapes) of these ligands. Such rigid analogues may be useful in deducing the bioactive conformation of these endogenous cannabinoids, not only at the CB receptors but also at the FAAH enzyme active site and possibly at the binding site(s) on the newly proposed anandamide transporter.

Arachidonic Acids↗

PubMed2001

Cannabinoid CB1-receptor mediated regulation of gastrointestinal motility in mice in a model of intestinal inflammation.

1. We have studied the effect of cannabinoid agonists (CP 55,940 and cannabinol) on intestinal motility in a model of intestinal inflammation (induced by oral croton oil in mice) and measured cannabinoid receptor expression, endocannabinoids (anandamide and 2-arachidonylglycerol) and anandamide amidohydrolase activity both in physiological and pathophysiological states. 2. CP 55,940 (0.03 - 10 nmol mouse(-1)) and cannabinol (10 - 3000 nmol mouse(-1)) were more active in delaying intestinal motility in croton oil-treated mice than in control mice. These inhibitory effects were counteracted by the selective cannabinoid CB(1) receptor antagonist SR141716A (16 nmol mouse(-1)). SR141716A (1 - 300 nmol mouse(-1)), administered alone, increased intestinal motility to the same extent in both control and croton oil-treated mice. 3. Croton oil-induced intestinal inflammation was associated with an increased expression of CB(1) receptor, an unprecedented example of up-regulation of cannabinoid receptors during inflammation. 4. High levels of anandamide and 2-arachidonylglycerol were detected in the small intestine, although no differences were observed between control and croton oil-treated mice; by contrast anandamide amidohydrolase activity increased 2 fold in the inflamed small intestine. 5. It is concluded that inflammation of the gut increases the potency of cannabinoid agonists possibly by 'up-regulating' CB(1) receptor expression; in addition, endocannabinoids, whose turnover is increased in inflamed gut, might tonically inhibit intestinal motility.

Analgesics↗

PubMed1998

Site-directed mutagenesis of essential residues involved in the mechanism of bacterial glycosylasparaginase.

Flavobacterium glycosylasparaginase was cloned in an Escherichia coli expression system. Site-directed mutagenesis was performed at residues suggested to be important in the catalytic mechanism based on the crystal structure of the human enzyme and other biochemical studies. In vitro autoproteolysis allowed the mutant enzymes to be activated, including those that were slow to self-cleave. Based on the activity of the mutant enzymes, six catalytically essential amino acids were identified: Trp-11, Asp-66, Thr-152, Thr-170, Arg-180, and Asp-183. Kinetic analysis of each mutant further defined the function of these residues in substrate specificity and reaction rate. Mutagenesis of the N-terminal nucleophile residue Thr-152 confirmed the key function of its side-chain hydroxyl group. Partial activities of mutants T152S/C were in agreement with the general mechanism of N-terminal nucleophile (Ntn)-amidohydrolases. The side-chain hydroxyl of Thr-170 contributes to the reaction rate based on studies of mutants T170S/C/A. Residues Asp-183 and Arg-180 were found to H-bond, respectively, with the charged alpha-amino and alpha-carboxyl group of the substrate (Asn-GlcNAc). Mutants R180Q/L and D183E/N had greatly decreased substrate affinity and reduced reaction rates. Kinetic studies also showed that Trp-11 is involved in regulation of the enzyme reaction rate, contradictory to a previous suggestion that this residue is involved in substrate binding. Asp-66 is a new residue found to be important in enzyme activity. The overall active site structure involving these catalytic residues resembles the glutaminase domain of glucosamine 6-phosphate synthase, another member of the Ntn-amidohydrolase family of enzymes.

Amino Acid Substitution↗

PubMed2004

Soybean cultivars 'Williams 82' and 'Maple Arrow' produce both urea and ammonia during ureide degradation.

The ability of two soybean (Glycine max L. [Merrill]) cultivars, 'Williams 82' and 'Maple Arrow', which were reported to use different ureide degradation pathways, to degrade the ureides allantoin and allantoate was investigated. Protein fractions and total leaf homogenates from the fourth trifoliate leaves of both cultivars were examined for the ability to evolve either (14)CO(2) or [(14)C]urea from (14)C-labelled ureides in the presence of various inhibitors. (14)CO(2) evolution from [2,7-(14)C]allantoate was catalysed by 25-50% saturated ammonium sulphate fractions of both cultivars. This activity was inhibited by acetohydroxamate (AHA), which has been used to inhibit plant ureases, but not by phenylphosphorodiamidate (PPD), a more specific urease inhibitor. Thus, in both cultivars, allantoate may be metabolized by allantoate amidohydrolase. This activity was sensitive to EDTA, consistent with previous reports demonstrating that allantoate amidohydrolase requires manganese for full activity. Total leaf homogenates of both cultivars evolved both (14)CO(2) and [(14)C]urea from [2,7-(14)C] (ureido carbon labelled) allantoin, not previously reported in either 'Williams 82' or in 'Maple Arrow'. In situ leaf degradation of (14)C-labelled allantoin confirmed that both urea and CO(2)/NH(3) are direct products of ureide degradation. Growth of plants in the presence of PPD under fixing and non-fixing conditions caused urea accumulation in both cultivars, but did not have a significant impact on total seed nitrogen. Urea levels were higher in N-fixing plants of both cultivars. Contrary to previous reports, no significant biochemical difference was found in the ability of these two cultivars to degrade ureides under the conditions used.

Allantoin↗

PubMed1999

N-Acylethanolamines in seeds. Quantification Of molecular species and their degradation upon imbibition

N-Acylethanolamines (NAEs) were quantified in seeds of several plant species and several cultivated varieties of a single species (cotton [Gossypium hirstutum]) by gas chromatography-mass spectroscopy. The total NAE content of dry seeds ranged from 490 +/- 89 ng g(-1) fresh weight in pea (Pisum sativum cv early Alaska) to 1,608 +/- 309 ng g(-1) fresh weight in cotton (cv Stoneville 7A glandless). Molecular species of NAEs in all seeds contained predominantly 16C and 18C fatty acids, with N-linoleoylethanolamine (NAE18:2) being the most abundant (approaching 1,000 ng g(-1) fresh weight in cottonseeds). Total NAE levels dropped drastically following 4 h of imbibition in seeds of pea, cotton, and peanut (Arachis hypogea cv Virginia), and this decline was most pronounced for NAE18:2. A novel enzyme activity was identified in cytosolic fractions of imbibed cottonseeds that hydrolyzed NAE18:2 in vitro. NAE degradation was optimal at 35 degrees C in 50 mM MES buffer, pH 6.5, and was inhibited by phenylmethylsulfonyl fluoride and 5, 5'-dithio-bis(2-nitrobenzoic acid), which is typical of other amide hydrolases. Amidohydrolase activity in cytosolic fractions exhibited saturation kinetics toward the NAE18:2 substrate, with an apparent K(m) of 65 &mgr;M and a V(max) of 83 nmol min(-1) mg(-1) protein. Total NAE amidohydrolase activity increased during seed imbibition, with the highest levels (about four times that in dry seeds) measured 2 h after commencing hydration. NAEs belong to the family of "endocannabinoids," which have been identified as potent lipid mediators in other types of eukaryotic cells. This raises the possibility that their imbibition-induced metabolism in plants is involved in the regulation of seed germination.

Journal Article↗

PubMed2002

The crystal structure of hypothetical protein MTH1491 from Methanobacterium thermoautotrophicum.

As part of our structural proteomics initiative, we have determined the crystal structure of MTH1491, a previously uncharacterized hypothetical protein from Methanobacterium thermoautotrophicum. MTH1491 is one of numerous structural genomics targets selected in a genome-wide survey of uncharacterized proteins. It belongs to a family of proteins whose biological function is not known. The crystal structure of MTH1491, the first structure for this family of proteins, consists of an overall five-stranded parallel beta-sheet with strand order 51234 and flanking helices. The oligomeric form of this molecule is a trimer as seen from both crystal contacts and gel filtration studies. Analysis revealed that the structure of MTH1491 is similar to that of dehydrogenases, amidohydrolases, and oxidoreductases. Using a combination of sequence and structural analyses, we showed that MTH1491 does not belong to either the dehydrogenase or the amidohydrolase superfamilies of proteins.

Bacterial Proteins↗

PubMed1988

Organization and multiple regulation of histidine utilization genes in Pseudomonas putida.

The arrangement of the histidine utilization (hut) genes in Pseudomonas putida was established by examining the structure of a DNA segment that had been cloned into Escherichia coli via a cosmid vector. Southern blot analysis revealed that the restriction patterns of the hut genes cloned into E. coli and present in the P. putida genome were identical, indicating that no detectable DNA rearrangement took place during the cloning. Expression of the hut genes from a series of overlapping clones indicated the gene order to be hutG-hutI-hutH-hutU-hutC-hutF. The transcription directions of the different hut genes were determined by cloning the genes under control of the lambda pL promoter. This showed that hutF, encoding formiminoglutamate hydrolase, was transcribed in a direction opposite to that of the other genes. Inactivation of the cloned hut genes by Tn1000 insertion revealed that the hut genes were divided into three major transcriptional units (hutF, hutC [the repressor gene], and hut UHIG), but hutG may also be independently transcribed. When cloned individually with hutC on the same vector, hutF and hutU (which encodes urocanase) expression was induced by urocanate, indicating that these two genes each possess an operator-promoter element. Tn1000 insertions (in the cloned genes) or Tn5 insertions (in the P. putida genome) affecting the hutI or hutH gene only partially eliminated hutG expression. Furthermore, hutG, which specifies N-formylglutamate amidohydrolase, was regulated by the hutC product when the two genes were cloned on the same vector and expressed in E. coli. Therefore, hutG can be expressed independently from its own promoter, in keeping with earlier observations that N-formylglutamate amidohydrolase synthesis is not coordinated with that of urocanase and histidase and can be induced by N-formylglutamate or urocanate.

Cloning, Molecular↗