Cysteine 180 Is a Redox Sensor Modulating the Activity of Human Pyridoxal 5'-Phosphate Histidine Decarboxylase.

Histidine decarboxylase is a pyridoxal 5'-phosphate enzyme catalyzing the conversion of histidine to histamine, a bioactive molecule exerting its role in many modulatory processes. The human enzyme is involved in many physiological functions, such as neurotransmission, gastrointestinal track function, cell growth, and differentiation. Here, we studied the functional properties of the human enzyme and, in particular, the effects exerted at the protein level by two cysteine residues: Cys-180 and Cys-418. Surprisingly, the enzyme exists in an equilibrium between a reduced and an oxidized form whose extent depends on the redox state of Cys-180. Moreover, we determined that (i) the two enzymatic redox species exhibit modest structural changes in the coenzyme microenvironment and (ii) the oxidized form is slightly more active and stable than the reduced one. These data are consistent with the model proposed by bioinformatics analyses and molecular dynamics simulations in which the Cys-180 redox state could be responsible for a structural transition affecting the C-terminal domain reorientation leading to active site alterations. Furthermore, the biochemical properties of the purified C180S and C418S variants reveal that C180S behaves like the reduced form of the wild-type enzyme, while C418S is sensitive to reductants like the wild-type enzyme, thus allowing the identification of Cys-180 as the redox sensitive switch. On the other hand, Cys-418 appears to be a residue involved in aggregation propensity. A possible role for Cys-180 as a regulatory switch in response to different cellular redox conditions could be suggested.

Effect of chemico-physical parameters on the histidine decarboxylase (HdcA) enzymatic activity in Streptococcus thermophilus PRI60.

UNLABELLED: In this study the activity of the histidine decarboxylase (HdcA) of Streptococcus thermophilus PRI60 was determined during growth and in crude enzyme preparations to evaluate its hazardousness in dairy products. The effect of different pH values, lactose availability, NaCl concentration, and growth temperature on histamine production was evaluated in M17 medium during 168 h incubation. In each case, the production of histamine increased concomitantly with the cell number with a relatively small further rise during the stationary phase. In all cultures the maximum histamine levels were reached at the end of active growth. Histamine was detectable (10 to 55 mg/L) even when growth was strongly inhibited. The HdcA enzyme in crude cell-free extracts was mostly active at acidic pH values common in dairy products. NaCl concentrations lower than 5% did not affect its activity. The enzyme was quite resistant to heat treatments resembling low pasteurization, but was inactivated at 75 °C for 2 min. Given the features of the enzyme studied, efforts must be dedicated to a thorough risk analysis and development of strategies to contrast the presence of histaminogenic S. thermophilus strains in products from raw or mildly heat-treated milk. PRACTICAL APPLICATION: During its growth Streptococcus thermophilus can produce histamine over a wide range of conditions encountered in cheesemaking and cheese ripening. The histidine-decarboxylase is even more active in cell-free extract and histamine can be accumulated independently of cell viability.

Histamine derived from probiotic Lactobacillus reuteri suppresses TNF via modulation of PKA and ERK signaling.

Beneficial microbes and probiotic species, such as Lactobacillus reuteri, produce biologically active compounds that can modulate host mucosal immunity. Previously, immunomodulatory factors secreted by L. reuteri ATCC PTA 6475 were unknown. A combined metabolomics and bacterial genetics strategy was utilized to identify small compound(s) produced by L. reuteri that were TNF-inhibitory. Hydrophilic interaction liquid chromatography-high performance liquid chromatography (HILIC-HPLC) separation isolated TNF-inhibitory compounds, and HILIC-HPLC fraction composition was determined by NMR and mass spectrometry analyses. Histamine was identified and quantified in TNF-inhibitory HILIC-HPLC fractions. Histamine is produced from L-histidine via histidine decarboxylase by some fermentative bacteria including lactobacilli. Targeted mutagenesis of each gene present in the histidine decarboxylase gene cluster in L. reuteri 6475 demonstrated the involvement of histidine decarboxylase pyruvoyl type A (hdcA), histidine/histamine antiporter (hdcP), and hdcB in production of the TNF-inhibitory factor. The mechanism of TNF inhibition by L. reuteri-derived histamine was investigated using Toll-like receptor 2 (TLR2)-activated human monocytoid cells. Bacterial histamine suppressed TNF production via activation of the H(2) receptor. Histamine from L. reuteri 6475 stimulated increased levels of cAMP, which inhibited downstream MEK/ERK MAPK signaling via protein kinase A (PKA) and resulted in suppression of TNF production by transcriptional regulation. In summary, a component of the gut microbiome, L. reuteri, is able to convert a dietary component, L-histidine, into an immunoregulatory signal, histamine, which suppresses pro-inflammatory TNF production. The identification of bacterial bioactive metabolites and their corresponding mechanisms of action with respect to immunomodulation may lead to improved anti-inflammatory strategies for chronic immune-mediated diseases.

Structural perspective on the direct inhibition mechanism of EGCG on mammalian histidine decarboxylase and DOPA decarboxylase.

Histidine decarboxylase (HDC) and l-aromatic amino acid decarboxylase (DDC) are homologous enzymes that are responsible for the synthesis of important neuroactive amines related to inflammatory, neurodegenerative, and neoplastic diseases. Epigallocatechin-3-gallate (EGCG), the most abundant catechin in green tea, has been shown to target histamine-producing cells and to promote anti-inflammatory, antitumor, and antiangiogenic effects. Previous experimental work has demonstrated that EGCG has a direct inhibitory effect on both HDC and DDC. In this study, we investigated the binding modes of EGCG to HDC and DDC as a first step for designing new polyphenol-based HDC/DDC-specific inhibitors.

A novel approach to inhibit intracellular vitamin B6-dependent enzymes: proof of principle with human and plasmodium ornithine decarboxylase and human histidine decarboxylase.

Pyridoxal-5'-phosphate (vitamin B(6))-dependent enzymes play central roles in the metabolism of amino acids. Moreover, the synthesis of polyamines, which are essential for cell growth, and of biogenic amines, such as histamine and other signal transmitters, relies on these enzymes. Certain B(6) enzymes thus are prime targets for pharmacotherapeutic intervention. We have devised a novel, in principle generally applicable strategy for obtaining small-molecule cell-permeant inhibitors of specific B(6) enzymes. The imine adduct of pyridoxal-5'-phosphate and the specific amino acid substrate, the first intermediate in all pyridoxal-5'-phosphate-dependent reactions of amino acids, was reduced to a stable secondary amine. This coenzyme-substrate-conjugate was modified further to make it membrane-permeant and, guided by structure-based modeling, to boost its affinity to the apoform of the target enzyme. Inhibitors of this type effectively decreased the respective intracellular enzymatic activity (IC(50) in low micromolar range), providing lead compounds for inhibitors of human ornithine decarboxylase (hODC), plasmodium ornithine decarboxylase, and human histidine decarboxylase. The inhibitors of hODC interfere with the metabolism of polyamines and efficiently prevent the proliferation of tumor cell lines (IC(50)∼ 25 µM). This approach to specific inhibition of intracellular B(6) enzymes might be applied in a straightforward manner to other B(6) enzymes of emerging medicinal interest.

Inhibitory and structural studies of novel coenzyme-substrate analogs of human histidine decarboxylase.

Histamine, a biogenic amine with important biological functions, is produced from histidine by histidine decarboxylase (HDC), a pyridoxal 5'-phosphate-dependent enzyme. HDC is thus a potential target to attenuate histamine production in certain pathological states. Targeting mammalian HDC with novel inhibitors and elucidating the structural basis of their specificity for HDC are challenging tasks, because the three-dimensional structure of mammalian HDC is still unknown. In the present study, we designed, synthesized, and tested potentially membrane-permeable pyridoxyl-substrate conjugates as inhibitors for human (h) HDC and modeled an active site of hHDC, which is compatible with the experimental data. The most potent inhibitory compound among nine tested structural variants was the pyridoxyl-histidine methyl ester conjugate (PHME), indicating that the binding site of hHDC does not tolerate groups other than the imidazole side chain of histidine. PHME inhibited 60% of the fraction of 12-O-tetradecanoylphorbol-13-acetate-induced newly synthesized HDC in human HMC-1 cells at 200 microM and was also inhibitory in cell extracts. The proposed model of hHDC, containing phosphopyridoxyl-histidine in the active site, revealed the binding specificity of HDC toward its substrate and the structure-activity relationship of the designed and investigated compounds.

Involvement of Sp1 in lipopolysaccharide-induced expression of HDC mRNA in RAW 264 cells.

The involvement of Sp1 in the lipopolysaccharide (LPS)-induced transcription of HDC mRNA in the mouse macrophage-like cell line RAW 264 was analyzed. LPS increased the levels of HDC mRNA 4 h after the stimulation in a concentration-dependent manner. Mithramycin A, an inhibitor of the binding of the Sp family to the GC box, reduced the LPS-induced increase in the levels of HDC mRNA at 4 h and HDC protein at 8 h in a concentration-dependent manner. By conducting electrophoretic mobility shift assays, we found that one of the transcription factors binding to the DNA probe containing the GC box sequence of the mouse HDC gene promoter region was Sp1, and that levels of Sp1-DNA probe complexes were increased by stimulation with LPS although the protein levels of Sp1 were not changed. These results suggested that Sp1 is one of the transcription factors that regulate the LPS-induced expression of HDC in RAW 264 cells.

Mammalian histidine decarboxylase: from structure to function.

Histamine is a multifunctional biogenic amine with relevant roles in intercellular communication, inflammatory processes and highly prevalent pathologies. Histamine biosynthesis depends on a single decarboxylation step, carried out by a PLP-dependent histidine decarboxylase activity (EC 4.1.1.22), an enzyme that still remains to be fully characterized. Nevertheless, during the last few years, important advances have been made in this field, including the generation and validation of the first three-dimensional model of the enzyme, which allows us to revisit previous results and conclusions. This essay provides a comprehensive review of the current knowledge of the structural and functional characteristics of mammalian histidine decarboxylase.

Mapping of catalytically important residues in the rat L-histidine decarboxylase enzyme using bioinformatic and site-directed mutagenesis approaches.

HDC (L-histidine decarboxylase), the enzyme responsible for the catalytic production of histamine from L-histidine, belongs to an evolutionarily conserved family of vitamin B6-dependent enzymes known as the group II decarboxylases. Yet despite the obvious importance of histamine, mammalian HDC enzymes remain poorly characterized at both the biochemical and structural levels. By comparison with the recently described crystal structure of the homologous enzyme L-DOPA decarboxylase, we have been able to identify a number of conserved domains and motifs that are important also for HDC catalysis. This includes residues that were proposed to mediate events within the active site, and HDC proteins carrying mutations in these residues were inactive when expressed in reticulocyte cell lysates reactions. Our studies also suggest that a significant change in quartenary structure occurs during catalysis. This involves a protease sensitive loop, and incubating recombinant HDC with an L-histidine substrate analogue altered enzyme structure so that the loop was no longer exposed for tryptic proteolysis. In total, 27 mutant proteins were used to test the proposed importance of 34 different amino acid residues. This is the most extensive mutagenesis study yet to identify catalytically important residues in a mammalian HDC protein sequence and it provides a number of novel insights into the mechanism of histamine biosynthesis.

The production of 53-55-kDa isoforms is not required for rat L-histidine decarboxylase activity.

Post-translational processing of the histamine-producing enzyme, L-histidine decarboxylase (HDC), leads to the formation of multiple carboxyl-truncated isoforms. Nevertheless, it has been widely reported that the mature catalytically active dimer is dependent specifically on the production of carboxyl-truncated 53-55-kDa monomers. Here we use transiently transfected COS-7 cells to study the properties of carboxyl-truncated rat HDC isoforms in the 52-58-kDa size range. Amino acid sequences important for the production of a 55-kDa HDC isoform were identified by successive truncations through amino acids 502, 503, and 504. Mutating this sequence in the full-length protein prevented the production of 55-kDa HDC but did not affect enzymatic activity. Further truncations to amino acid 472 generated an inactive 53-kDa HDC isoform that was degraded by the proteasome pathway. These results suggested that processed isoforms, apart from 53-55-kDa ones, contribute toward histamine biosynthesis in vivo. This was confirmed in physiological studies where regulated increases in HDC activity were associated with the expression of isoforms that were greater than 55 kDa in size. We provide evidence to show that regulation of HDC expression can be achieved by the differential production or differential stabilization of multiple enzyme isoforms.

The pest regions containing C-termini of mammalian ornithine decarboxylase and histidine decarboxylase play different roles in protein degradation.

Proteasome 26S must recognize the PEST region-containing C-terminus of mammalian ornithine decarboxylase (ODC) monomer to proceed with degradation. We have detected PEST regions in both termini of mammalian histidine decarboxylase (HDC). In the present report, a chimaeric ODC/HDC was used to elucidate whether the PEST region-containing C-termini of ODC and HDC are exchangeable. Wild-type rat ODC had an expected antizyme and ATP-dependent degradation. This was not the case for both the chimaera and a C-terminus truncated rat ODC. Results suggest that the PEST region-containing C-terminus of rat HDC should have another role different to confering polypeptide availability to the proteasome.

De Novo synthesis and posttranslational processing of L-histidine decarboxylase in mice.

We purified L-histidine decarboxylase from mouse mastocytoma cells and cloned mouse HDC cDNA, and found that the primary translated product (74 kD) is posttranslationally processed in its C-terminal region to yield a native HDC subunit (53 kD). Recombinant 74-kD, but not 53-kD HDC species was present mainly in the particulate fraction of Sf9 cells. The particulate 74-kD recombinant HDC was cleaved by porcine pancreatic elastase, and a homodimer of a 53-kD subunit having the identical catalytic properties to those of native HDC was solubilized. The particulate HDC from mouse stomach was partially purified and it was solubilized by porcine pancreatic elastase to yield the 53-kD subunit of HDC. We identified endogenous proteolytic activity, which converts the particulate recombinant 74-kD HDC to the soluble 53-kD HDC in the supernatant of mouse stomach. In mastocytoma cells, we demonstrated that the induction of HDC activity and HDC mRNA synergistically occurred upon treatment with dexamethasone + TPA, and also with cAMP + Ca2+. On a genomic DNA cloning, we found that two upregulations occurred via the involvement of the regulatory elements binding to the sequences from -132 to -53 and -267 to -53, respectively.

Processing and activation of recombinant mouse mastocytoma histidine decarboxylase in the particulate fraction of Sf9 cells by porcine pancreatic elastase.

Mature 53 kDa histidine decarboxylase (HDC) peptide is produced from a precursor 74 kDa peptide. The mechanism of specific cleavage by processing enzyme is unknown. Using the recombinant mouse 74 kDa HDC, we found that porcine pancreatic elastase specifically converted the inactive 74 kDa HDC to its active form of 53 kDa HDC.

A histidine decarboxylase gene encoded by the Vibrio anguillarum plasmid pJM1 is essential for virulence: histamine is a precursor in the biosynthesis of anguibactin.

We have identified and sequenced an hdc gene in the Vibrio anguillarum plasmid pJM1 which encodes a histidine decarboxylase enzyme and is an essential component for the biosynthesis of anguibactin. The open reading frame corresponds to a protein of 386 amino acids with a calculated molecular mass of 44,259.69 Da. The amino acid sequence has extensive homology with the pyridoxal-P-dependent histidine decarboxylases of Morganella morganii, Klebsiella planticola, and Enterobacter aerogenes. Tn3-HoHo1 transposition mutagenesis of the hdc gene present in a recombinant clone carrying the entire pJM1 iron uptake region produced two derivatives, one with the lacZ gene in the same orientation as the direction of hdc transcription and the other with the lacZ gene in the opposite orientation. A. V. anguillarum strain harbouring one of the mutated derivatives was unable to grow under iron-limiting conditions and did not produce anguibactin. Therefore, the hdc gene must play a role in the biosynthetic pathway of this siderophore and consequently in conferring the high virulence phenotype to this bacterium. The role of histidine decarboxylase in biosynthesis of anguibactin was confirmed by the fact that growth under iron starvation was restored by addition of histamine to the medium. The presence of anguibactin was also demonstrated in supernatants from cultures of the hdc mutant strains grown under iron starvation with the addition of histamine, further confirming that histamine is a precursor in the biosynthesis of the siderophore.(ABSTRACT TRUNCATED AT 250 WORDS)

[Molecular biology of L-histidine decarboxylase].

L-Histidine decarboxylase (HDC) catalyzes the formation of histamine from L-histidine. This biogenic amine is known to exert various effects in physiological and pathological reactions. In contrast to the well-known mechanism of histamine action through its interaction with specific receptors, the mechanisms regulating HDC gene expression are not elucidated. We have purified HDC from mouse mastocytoma cells, and isolated mouse HDC cDNA, and found that the primary translated product is posttranslationally processed to yield a mature active enzyme. In mastocytoma cells, we demonstrated that the induction of HDC activity and HDC mRNA synergistically occurred on treatment with dexamethasone+TPA, and also cAMP+Ca2+. To clarify the mechanism of up-regulation by these stimuli of the transcription of the HDC gene, we have isolated a genomic DNA clone encoding 5'-flanking region sequence and the first two exons. The transcription start site and the nucleotide sequences of the promoter regions including TATA- and GC-boxes were determined. With mastocytoma cells transiently transfected with 5' deletion constructs of HDC-CAT fusion gene, it was found that the sequences from -132 to -53 and -267 to -53 are essential for the regulatory elements involved in the increased transcription of the HDC gene with dexamethasone+TPA and cAMP+Ca2+, respectively. Furthermore, we have isolated a genomic DNA from human basophilic cells, and analysed its structure to elucidate the mechanisms regulating the tissue specificity of HDC gene expression.

Purification and properties of L-histidine decarboxylase from mouse stomach.

L-Histidine decarboxylase was purified to electrophoretic homogeneity from mouse stomach according to a procedure described previously [Ohmori E, Fukui T, Imanishi N, Yatsunami K and Ichikawa A, J Biochem (Tokyo) 107: 834-839, 1990]. The purified enzyme exhibited a specific activity of 750 nmol histamine formed per min per mg protein, which constituted a 37,500-fold purification compared to the crude extract, with a 1.6% yield. The molecular mass of the enzyme was estimated to be 54 kDa by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 100 kDa by gel filtration. The isoelectric point of the enzyme was determined to be pH 5.4. The Km value for L-histidine was estimated to be 0.29 mM. The single mRNA encoding the amino acid sequence of the mouse stomach enzyme was examined and its size was found to be 2.7 kb. These molecular and catalytic property values of the L-histidine decarboxylase of mouse stomach are quite similar to those of the enzyme from mouse mastocytoma P-815 cells.