Production of recombinant rat hepatic histidase.

Rat liver histidase was expressed in E. coli by using a PCR product of the coding sequence obtained from the rat liver cDNA of histidase cloned in the expression vector pRSET. The construct (pRSET-HAL) produced a fusion protein containing a tail of polyhistidine. The expression product was purified with a resin containing Ni+ that retains proteins with polyhistidine fragments. pRSET-HAL was analyzed by restriction enzyme mapping and by sequencing confirming the correct orientation and nucleotide sequence. Native rat liver histidase was also purified and it had a Mr of 72 kDa. An antiserum against native histidase was obtained in rabbit. Western blot analysis revealed one band of 72 kDa observed in membranes containing purified histidase or rat liver high speed supernatants. The same antiserum also detected in cell lysates of E. coli transformed with the pRSET-HAL plasmid a single band of 74 kDa of the recombinant histidase before cleavage with enterokinase. After the proteolysis, the Western blot analysis showed a single band of approximately 72 kDa. Kinetic analysis of the recombinant histidase showed similar Km and Vmax compared with native histidase.

Production of recombinant rat hepatic histidase / Producción de histidasa recombinante de hígado de rata

Rat liver histidase was expressed in E. coli by using a PCR product of the coding sequence obtained from the rat liver cDNA of histidase cloned in the expression vector pRSET. The construct (pRSET-HAL) produced a fusion protein containing a tail of polyhistidine. The expression product was purified with a resin containing Ni+ that retains proteins with polyhistidine fragments. pRSET-HAL was analyzed by restriction enzyme mapping and by sequencing confirming the correct orientation and nucleotide sequence. Native rat liver histidase was also purified and it had a Mr of 72 kDa. An antiserum against native histidase was obtained in rabbit. Western blot analysis revealed one band of 72 kDa observed in membranes containing purified histidase or rat liver high speed supernatants. The same antiserum also detected in cell lysates of E. coli transformed with the pRSET-HAL plasmid a single band of 74 kDa of the recombinant histidase before cleavage with enterokinase. After the proteolysis, the Western blot analysis showed a single band of approximately 72 kDa. Kinetic analysis of the recombinant histidase showed similar Km and Vmax compared with native histidase.

La histidasa de hígado de rata se expresó a partir del producto de amplificación por PCR de la región codificante del DNAc de la histidasa, el cual se clonó en el vector de expresión pRSET. La construcción (pRSET-HAL) permitió la producción de una proteína de fusión que contenía una cola de polihistidinas. El producto de expresión se purificó con una resina que contiene Ni+, la cual retiene proteínas con fragmentos de polihistidina. El pRSET-HAL se analizó por medio de un análisis con enzimas de restricción y por secuenciación de DNA para confirmar su correcta orientación así como tambièn su secuencia nucleotídica. La histidasa nativa hepática fue tambièn purificada y tuvo un Mr de 72 kDa. A partir de la enzima purificada nativa se preparó un antisuero contra la histidasa en conejo. El análisis por Western blot mostró una sola banda de 72 kDa en membranas que contenían histidasa purificada o en la fracción citosólica de hígado de rata. El mismo anticuerpo tambièn detectó en lisados de E. coli transformados con el plásmido pRSET-HAL una sola banda de 74 kDa correspondientes a la histidasa recombinante antes de su hidrólisis con enterocinasa. Despuès de la proteólisis, el análisis por Western blot mostró una sola banda de aproximadamente 72 kDa correspondiente a la histidasa. Análisis cinèticos de la histidasa recombinante mostraron Km y Vmax similares a las observadas con la histidasa nativa.

Crystallization and preliminary X-ray studies of Pseudomonas putida histidine ammonium-lyase.

Histidine ammonium-lyase from P. putida was expressed in Escherichia coli, purified to homogeneity, and crystallized by the vapour-diffusion method using polyethylene glycol 3350 as the precipitant. The crystals, which diffract to at least 2.5 A resolution, exhibit the symmetry of space group P212121, with unit-cell parameters a = 89.7, b = 138.2 and c = 164.8 A. The asymmetric unit contains a tetramer, and the crystals have a Vm value of 2.41 A3 Da-1.

Identification of serine-143 as the most likely precursor of dehydroalanine in the active site of histidine ammonia-lyase. A study of the overexpressed enzyme by site-directed mutagenesis.

The gene coding for histidase (histidine ammonia-lyase, HAL, EC 4.3.1.3) was isolated from a lambda-EMBL3 genomic library from Pseudomonas putida nicII and subcloned into the expression vector pT7-7. Transformation of Escherichia coli BL21 (DE3) cells with the recombinant vector led to the expression of catalytically active histidase amounting to 20-30% of the total soluble protein in the crude cell extract. A new rapid and highly efficient isolation procedure is described leading to electrophoretically homogeneous histidase within 1.5 days. Six grams of E. coli BL21 (DE3) cells (wet weight) gives approximately 100 mg of homogeneous histidase with a specific activity of 27 IU/mg. To investigate the possible role of serine as a precursor of dehydroalanine in the active site of histidase, each of the four serines, conserved in all known histidases and phenylalanine ammonia-lyases, was consecutively changed to alanine by site-directed mutagenesis. The resulting mutant genes were subcloned into the expression vector pT7-7 and were assayed for histidase activity. The catalytic activities of the four mutants and of wild-type histidase were compared. The Km and Vmax values of the overexpressed mutants S112A, S393A, and S418A and wild-type histidase did not show any significant differences. Mutant S143A, however, was devoid of catalytic activity (< 0.01%), pointing to the outstanding importance of this serine for the formation of an active enzyme. We conclude that serine-143 is the most probable precursor of the active-site dehydroalanine. The role of serine-143 in the biosynthesis of active histidase is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of Pseudomonas putida histidine ammonia-lyase expressed in Escherichia coli.

Histidine ammonia-lyase (HAL) from Pseudomonas putida PRS1 contains a catalytically important electrophilic center reported to be dehydroalanine. Little is known about the origin of this group or its linkage to the protein. To initiate structural studies on this enzyme, P. putida HAL was purified from an Escherichia coli high-expression clone in which the HAL gene (hutH) was under the control of the lambda PL promoter on a plasmid vector. In this clone from 6 to 10% of the soluble cell protein after heat induction was HAL and approximately 200 mg of 95% pure HAL could be obtained from 120 g wet weight of cells in a 40 to 60% yield. The overexpressed protein was identical to P. putida HAL in native molecular weight (220 kDa), subunit composition (four identical subunits of 53 kDa each), affinity for substrate (L-histidine Km of 5.3 mM at pH 9.0), and its sensitivity to inactivation by cyanide and bisulfite. The N-terminal amino acid sequence was in agreement with the DNA-predicted sequence, indicating proper translational initiation. These features make this enzyme an appropriate candidate for protein structure investigations regarding the nature of the electrophilic center and its association with the protein.

Histidine ammonia-lyase from Streptomyces griseus.

Histidine ammonia-lyase (histidase; HutH) has been purified to homogeneity from Streptomyces griseus and the N-terminal amino acid (aa) sequence used to clone the histidase-encoding structural gene, hutH. The purified enzyme shows typical saturation kinetics and is inhibited competitively by D-histidine and histidinol phosphate. High concentrations of K.cyanide inactivate HutH unless the enzyme is protected by the substrate or histidinol phosphate. On the basis of the nucleotide sequence, the hutH structural gene would encode a protein of 53 kDa with an N terminus identical to that determined for the purified enzyme. Immediately upstream from hutH is a region that strongly resembles a class of Streptomyces promoters active during vegetative growth; however, there is no obvious ribosome-binding site adjacent to the hutH translation start codon. The deduced aa sequence of an upstream partial open reading frame shows no similarity with other proteins, including HutP of Bacillus subtilis and HutU of Pseudomonas putida. Promoter-probe analysis indicates that promoter activity maps within the DNA surrounding the hutH start codon. Pairwise comparisons of the primary structures of bacterial and mammalian histidases, together with the unique kinetic properties and gene organization, suggest that streptomycete histidase may represent a distinct family of histidases.

Purification of histidase from Streptomyces griseus and nucleotide sequence of the hutH structural gene.

Histidine ammonia-lyase (histidase) was purified to homogeneity from vegetative mycelia of Streptomyces griseus. The enzyme was specific for L-histidine and showed no activity against the substrate analog, D-histidine. Histidinol phosphate was a potent competitive inhibitor. Histidase displayed saturation kinetics with no detectable sigmoidal response. Neither thiol reagents nor a variety of divalent cations had any effect on the activity of the purified enzyme. High concentrations of potassium cyanide inactivated histidase in the absence of its substrate or histidinol phosphate, suggesting that, as in other histidases, dehydroalanine plays an important role in catalysis. The N-terminal amino acid sequence of histidase was used to construct a mixed oligonucleotide probe to identify and clone the histidase structural gene, hutH, from genomic DNA of the wild-type strain of S. griseus. The cloned DNA restored the ability of a histidase structural gene mutant to grow on L-histidine as the sole nitrogen source. The deduced amino acid sequence of hutH shows significant relatedness with histidase from bacteria and a mammal as well as phenylalanine ammonia-lyase from plants and fungi.

Cascading regulation of histidase activity in Streptomyces griseus.

Mutants of Streptomyces griseus unable to utilize histidine as the sole nitrogen source have been isolated and characterized. Using a mutant defective in the production of histidase, we have demonstrated that urocanate functions as the inducer of the histidine utilization system. Another mutant produced histidase that was locked in an inactive form but could be activated by treatment with an extract from the wild-type strain or the histidase-negative strain. This mutant was deficient in the activity of a protein of Mr ca. 90,000 to 100,000 that is required for the activation of histidase. Histidase was synthesized constitutively but was maintained in an inactive form until after histidine or urocanate was added to the medium. At least four components were implicated in the activation of histidase: histidase, the activation protein, urocanate, and a phosphatase that is apparently inactive in cells grown without inducer. The functions of the last three factors could be supplanted in vitro by incubation of histidase with snake venom phosphodiesterase or 5' nucleotidase. The results suggest that histidine utilization by S. griseus is controlled posttranslationally by an activation cascade that involves at least two regulatory proteins.

A comparative study of hepatic and epidermal histidase in the guinea-pig (Cavia porcellus).

Histidine ammonia lyase was purified to homogeneity from guinea-pig liver and epidermis. Both enzymes had similar molecular weights, subunit composition and pH optima. Km values for the two were similar at pH 9.2 but different at pH 7.0. Both enzymes were stimulated by low thiol concentrations and inhibited at higher concentrations, but to different extents. Antibody to the hepatic enzyme showed complete identity against hepatic enzyme but incomplete identity against epidermal enzyme.

The effect of histidine ammonia-lyase on some murine tumours.

The histidine ammonia-lyase from bacterial strain CAMR 5315 was partially purified to assess its effect on the growth of murine tumours. This strain was selected as the source after an extensive screening programme for histidine ammonia-lyases. The enzyme was partially purified by ammonium sulphate fractionation, chromatography on DEAE-cellulose and Sephadex G-150. The enzyme reduced circulating L-histidine levels in Wistar rats and in mice persisted with a half-life of 6-7 h. Neither LDH virus nor chemical modification with ethylacetimidate increased the half-life as observed with L-asparaginase and L-glutaminase. The enzyme was tested in mice against Ehrlich carcinoma, L5178Y lymphoblastic leukaemia, Mc/S sarcoma, B16 melanoma, P8157 mastocytoma, P1798 lymphosarcoma and the Gardner 6C3HED lymphosarcoma. The only tumours to show sensitivity to the enzyme were the Mc/S sarcoma against which a 65% increase in life span was observed at the highest enzyme dose, 1000 U/kg on alternate days over 14 days and the Ehrlich ascites carcinoma where cures were obtained at 250 U/kg on alternate days over 14 days, but only at inocula levels of 10(5) and 10(3) cells/animal respectively.

L-Histidine ammonia-lyase activity of axenically grown Hartmannella culbertsoni.

1. Histidine ammonia-lyase (EC 4.3.1.3) activity in the cell-free extracts of Hartmannella culbertsoni has been partially purified and the optimum activity is found at pH 9.0--9.2. 2. The enzyme required sulphydryl groups for its activity. L-2-Thiohistidine and EDTA competitively inhibit the enzyme. 3. Its molecular weight, as determined by gel filtration technique, is 131,800 daltons and the energy of activation for this enzyme is 15,205 cals/mole. 4. Certain amoebicidal drug and divalent cations have marked inhibitory effect on the enzyme. Co2+ has a profound stimulatory effect.

Crystalline L-histidine ammonia-lyase of Achromobacter liquidum. Crystallization and enzymic properties.

Crystalline L-histidine ammonia-lyase of Achromobacter liquidum was prepared with a 24% recovery of the activity. The specific activity of the pure enzyme (63 mumol of urocanic acid min-1 mg-1) is similar to those so far reported for the enzyme from other sources. The purified enzyme appeared to be homogeneous by analytical disc electrophoresis and isoelectric focusing (pI = 4.95). The molecular weight determined by Sephadex G-200 gel filtration is 200000. The optimum pH is 8.2, and the optimum temperature is 50 degrees C. The enzyme showed strict specificity to L-histidine (Km = 3.6 mM). Several histidine derivatives are not susceptible to the enzyme but do inhibit the enzyme activity competitively; the most effective inhibitors are L-histidine methyl ester (Ki = 3.66 mM) and beta-imidazole lactic acid (Ki = 3.84 mM). L-Histidine hydrazide (Ki = 36 mM) and imidazole (Ki = 6 mM) noncompetitively inhibited the enzyme EDTA markedly inhibited enzyme activity and this inhibition were reversed by divalent metal ions such as Mn2+, Co2+ Zn2+, Ni2+, Mg2+, and Ca2+. These results suggest that the presence of divalent metal ions is necessary for the catalytic activity of histidine ammonia-lyase. Sodium borohydride and hydrogen peroxide inhibited the enzyme activity.