An interaction network in Bacillus subtilis coproporphyrinogen oxidase is essential for the oxidation of protoporphyrinogen IX.

Coproporphyrinogen oxidase (CPO) plays important role in the biosynthesis of heme by catalyzing the coproporphyrinogen III to coproporphyrin III. However, in earlier research, it was regarded as the protoporphyrinogen oxidase (PPO) because it can also catalyze the oxidation of protoporphyrinogen IX to protoporphyrin IX. Identification of the commonalities in CPO and PPO would help us to get a further understanding of the enzyme function. In this work, we explored the role of a non-conserved residue, Asp65 in Bacillus subtilis CPO (bsCPO), whose corresponding residues in PPO from various species are neutral or positive residue (arginine in human PPO or asparagine in tobacco PPO, etc.). We found that Asp65 performs its function by forming a polar interaction network with its surrounding residues in bsCPO, which is important for enzymatic activity. This polar network maintains the substrate binding chamber and stabilizes the micro-environment of the isoalloxazine ring of FAD for the substrate-FAD interaction. Both the comparison of the crystal structures of bsCPO with PPO and our previous work showed that a similar polar interaction network is also present in PPOs. The results confirmed our conjecture that non-conserved residues can form a conserved element to maintain the function of CPO or PPO.

Recent advances in HemN-like radical S-adenosyl-l-methionine enzyme-catalyzed reactions.

Covering: 2012 to 2019HemN-like radical S-adenosyl-l-methionine (SAM) enzymes have been recently disclosed to catalyze diverse chemically challenging reactions from primary to secondary metabolic pathways. In this highlight, we summarize the reaction examples catalyzed by HemN-like enzymes to date and the enzymatic mechanisms reported. From the recent mechanistic investigations, we reason that there is a shared initiating mechanism wherein a characteristic SAM methylene radical is proposed to abstract a hydrogen atom from an sp3 carbon or add onto an sp2 carbon center although variations occur thereafter from reaction to reaction, as well as providing a brief insight into some future prospects.

Heme synthesis through the life cycle of the heme auxotrophic parasite Leishmania major.

Heme is an essential molecule synthetized through a broadly conserved 8-step route that has been lost in trypanosomatid parasites. Interestingly, Leishmania reacquired by horizontal gene transfer from γ-proteobacteria the genes coding for the last 3 enzymes of the pathway. Here we show that intracellular amastigotes of Leishmania major can scavenge heme precursors from the host cell to fulfill their heme requirements, demonstrating the functionality of this partial pathway. To dissect its role throughout the L. major life cycle, the significance of L. major ferrochelatase (LmFeCH), the terminal enzyme of the route, was evaluated. LmFeCH expression in a heterologous system demonstrated its activity. Knockout promastigotes lacking lmfech were not able to use the ferrochelatase substrate protoporphyrin IX as a source of heme. In vivo infection of Phlebotomus perniciosus with knockout promastigotes shows that LmFeCH is not required for their development in the sandfly. In contrast, the replication of intracellular amastigotes was hampered in vitro by the deletion of lmfech. However, LmFeCH-/- parasites produced disease in a cutaneous leishmaniasis murine model in a similar way as control parasites. Therefore, although L. major can synthesize de novo heme from macrophage precursors, this activity is dispensable being an unsuited target for leishmaniasis treatment.-Orrego, L. M., Cabello-Donayre, M., Vargas, P., Martínez-García, M., Sánchez, C., Pineda-Molina, E., Jiménez, M., Molina, R., Pérez-Victoria, J. M. Heme synthesis through the life cycle of the heme auxotrophic parasite Leishmania major.

Cystathionine-γ-lyase (CSE) deficiency increases erythropoiesis and promotes mitochondrial electron transport via the upregulation of coproporphyrinogen III oxidase and consequent stimulation of heme biosynthesis.

BACKGROUND: Hydrogen sulfide (H2S) is an endogenous gasotransmitter produced by mammalian cells. The current study investigated the potential role of H2S in the regulation of heme biosynthesis using mice deficient in cystathionine gamma-lyase (CSE), one of the three major mammalian H2S-producing enzymes. METHODS: Wild-type and global CSE-/- mice, as well as mitochondria prepared from their liver were used. In vivo, arterial and venous blood gases were measured, and survival of the mice to severe global hypoxia was monitored. Ex vivo, expression of various heme biosynthetic enzymes including coproporphyrinogen oxidase (CPOX) was measured, and mitochondrial function was evaluated using Extracellular Flux Analysis. Urine samples were collected to measure the oxidized porphyrinogen intermediates. The in vivo/ex vivo studies were complemented with mitochondrial bioenergetic studies in hepatocytes in vitro. Moreover, the potential effect of H2S on the CPOX promoter was studied in cells expressing a CPOX promoter construct system. RESULTS: The main findings are as follows: (1) CSE-/- mice exhibit elevated red blood cell counts and red blood cell mean corpuscular volumes compared to wild-type mice; (2) these changes are associated with elevated plasma and liver heme levels and (3) these alterations are likely due to an induction of CPOX (the sixth enzyme involved in heme biosynthesis) in CSE-/- mice. (4) Based on in vitro promoter data the promoter activation of CPOX is directly influenced by H2S, the product of CSE. With respect to the potential functional relevance of these findings, (5) the increased circulating red blood cell numbers do not correspond to any detectable alterations in blood gas parameters under resting conditions, (6) nor do they affect the hypoxia tolerance of the animals in an acute severe hypoxia model. However, there may be a functional interaction between the CSE system and the CPOX system in terms of mitochondrial bioenergetics: (7) CSE-/- hepatocytes and mitochondria isolated from them exhibit increased oxidative phosphorylation parameters, and (8) this increase is partially blunted after CPOX silencing. Although heme is essential for the biosynthesis of mitochondrial electron chain complexes, and CPOX is required for heme biosynthesis, (9) the observed functional mitochondrial alterations are not associated with detectable changes in mitochondrial electron transport chain protein expression. CONCLUSIONS: The CSE system regulates the expression of CPOX and consequent heme synthesis. These effects in turn, do not influence global oxygen transport parameters, but may regulate mitochondrial electron transport.

Revisiting the Mechanism of the Anaerobic Coproporphyrinogen†III Oxidase HemN.

HemN is a radical S-adenosyl-l-methionine (SAM) enzyme that catalyzes the oxidative decarboxylation of coproporphyrinogen III to produce protoporphyrinogen IX, an intermediate in heme biosynthesis. HemN binds two SAM molecules in the active site, but how these two SAMs are utilized for the sequential decarboxylation of the two propionate groups of coproporphyrinogen III remains largely elusive. Provided here is evidence showing that in HemN catalysis a SAM serves as a hydrogen relay which mediates a radical-based hydrogen transfer from the propionate to the 5'-deoxyadenosyl (dAdo) radical generated from another SAM in the active site. Also observed was an unexpected shunt product resulting from trapping of the SAM-based methylene radical by the vinyl moiety of the mono-decarboxylated intermediate, harderoporphyrinogen. These results suggest a major revision of the HemN mechanism and reveal a new paradigm of the radical-mediated hydrogen transfer in radical SAM enzymology.

Aerobic Enzymes and Their Radical SAM Enzyme Counterparts in Tetrapyrrole Pathways.

Microorganisms have lifestyles and metabolism adapted to environmental niches, which can be very broad or highly restricted. Molecular oxygen (O2) is currently variably present in microenvironments and has driven adaptation and microbial differentiation over the course of evolution on Earth. Obligate anaerobes use enzymes and cofactors susceptible to low levels of O2 and are restricted to O2-free environments, whereas aerobes typically take advantage of O2 as a reactant in many biochemical pathways and may require O2 for essential biochemical reactions. In this Perspective, we focus on analogous enzymes found in tetrapyrrole biosynthesis, modification, and degradation that are catalyzed by O2-sensitive radical S-adenosylmethionine (SAM) enzymes and by O2-dependent metalloenzymes. We showcase four transformations for which aerobic organisms use O2 as a cosubstrate but anaerobic organisms do not. These reactions include oxidative decarboxylation, methyl and methylene oxidation, ring formation, and ring cleavage. Furthermore, we highlight biochemically uncharacterized enzymes implicated in reactions that resemble those catalyzed by the parallel aerobic and anaerobic enzymes. Intriguingly, several of these reactions require insertion of an oxygen atom into the substrate, which in aerobic enzymes is facilitated by activation of O2 but in anaerobic organisms requires an alternative mechanism.

Hiding in the Shadows: CPOX Expression and 5-ALA Induced Fluorescence in Human Glioma Cells.

Protoporphyrin IX (PpIX) is widely used in photodynamic diagnosis. To date, the details of molecular mechanisms underlying PpIX accumulation in malignant cells after 5-ALA administration remain unclear. The fluorescence of PpIX was studied in human glioma cells. Several cell cultures were established from glioma tumor tissue to study the differences between fluorescence-positive and fluorescence-negative human glioma tumors. The cell cultures demonstrated fluorescence profiles similar to those of source tumor tissues, which allows us to use these cultures in experimental research. Dynamics of the rates of synthesis and degradation of fluorescent protoporphyrin IX was studied in the cultures obtained. In addition, the expression of CPOX, an enzyme involved in PpIX synthesis, was evaluated. mRNA levels of heme biosynthesis enzymes were analyzed, and PpIX fluorescence proved to correlate with the CPOX protein level, whereas no such correlation was observed at the mRNA level. Fluorescence intensity decreased at low levels of the enzyme, which indicates its critical role in PpIX fluorescence. Finally, the fluorescence intensity proved to correlate with the proliferative activity.

Calcitriol enhances the effect of photodynamic therapy in human breast cancer.

PURPOSE: To investigate the killing effect of photodynamic therapy (PDT) mediated by hematoporphyrin derivative (HPD) on human breast cancer MCF7 and MDA-MB-231 cells in vitro. METHODS: MCF7 and MDA-MB-231 breast cancer cells cultured in vitro were incubated with calcitriol (concentration of 10-8M, 10-10 M, 10-12 M, 10-14 M, 10-16 M, 0 M) to determine a proper concentration. The cells were divided into experimental group (calcitriol, HPD group and laser), HPD group (HPD and laser), calcitriol group (calcitriol and laser), blank laser group (laser alone) and blank group (no drugs and laser). Then the cells were preconditioned with calcitriol for 48 hrs and incubated with HPD for 6 hrs. After light exposure with 630 nm laser, the cells' viability and the reactive oxygen species (ROS) were assessed. After 8 hrs, flow cytometry was applied to detect the rate of cell apoptosis. The fluorescence intensity in cells was detected. Furthermore, the expression of porphyrin synthetic enzymes in pretreated breast cancer cells was analyzed. RESULTS: MTT assay showed that the viability of cells in the experimental group was lowest (p<0.05). The ROS intensity of the experimental group was higher (p<0.01). The rate of cell apoptosis was higher in the experimental group (p<0.05), and the fluorescence of the experimental group was higher (p<0.01). Furthermore, mechanistic studies documented that the expression of the porphyrin synthesis enzyme coproporphyrinogen oxidase (CPOX) was increased by calcitriol at the mRNA level. CONCLUSION: This research revealed a simple, non-toxic and highly effective preconditioning regimen to selectively enhance protoporphyrin IX (PpIX) fluorescence and the response of HPD-PDT in breast cancer search. This finding suggests that the combined treatment of breast cancer cells with calcitriol plus HPD may provide an effective and selective therapeutic modality to enhance HPD-induced PpIX fluorescent quality for improving discrimination of tumor tissue and PDT efficacy.

Role of HemF and HemN in the heme biosynthesis of Vibrio vulnificus under S-adenosylmethionine-limiting conditions.

Vibrio vulnificus contains two coproporphyrinogen III oxidases (CPOs): O2-dependent HemF and O2-independent HemN. The growth of the hemF mutant HF1 was similar to wild-type cells at pH 7.5 under 2% O2 conditions where HemN was active and had a half-life of 64 min. However, HF1 did not grow when the medium pH decreased to pH 5.0, where oxidative stress affects endogenous S-adenosylmethionine (SAM) levels. The growth of HF1 was restored not only by elevating the expression of MnSOD but also through the exogenous addition of SAM. For HF1 to grow under these SAM-limiting conditions, a mutation arose in hemN, encoding HemNY74F . Refolding of the denatured enzymes in vitro revealed that the apparent binding affinity of HemNY74F for the cofactor SAM1, which coordinates the 4Fe-4S cluster, was approximately sixfold higher than that of HemN. The Km of HemNY74F for the co-substrate SAM2, which provides radicals for CPO reactions, was threefold lower than that of HemN. Thus, affinities for both SAM1 and SAM2 were higher with the Y74F mutation. Taken together, when SAM is limiting, HemN is apparently nonfunctional, and heme synthesis is continued by HemF.

Oxygen-dependent copper toxicity: targets in the chlorophyll biosynthesis pathway identified in the copper efflux ATPase CopA deficient mutant.

Characterization of a copA(-) mutant in the purple photosynthetic bacterium Rubrivivax gelatinosus under low oxygen or anaerobic conditions, as well as in the human pathogen Neisseria gonorrhoeae identified HemN as a copper toxicity target enzyme in the porphyrin synthesis pathway. Heme synthesis is, however, unaffected by copper under high oxygen tension because of the aerobic coproporphyrinogen III oxidase HemF. Nevertheless, in the copA(-) mutant under aerobiosis, we show that the chlorophyll biosynthesis pathway is affected by excess copper resulting in a substantial decrease of the photosystem. Analyses of pigments and enzyme activity showed that under low copper concentrations, the mutant accumulated protochlorophyllide, suggesting that the protochlorophyllide reductase activity is affected by excess copper. Increase of copper concentration led to a complete lack of chlorophyll synthesis as a result of the loss of Mg-chelatase activity. Both enzymes are widely distributed from bacteria to plants; both are [4Fe-4S] proteins and oxygen sensitive; our data demonstrate their in vivo susceptibility to copper in the presence of oxygen. Additionally, our study provides the understanding of molecular mechanisms that may contribute to chlorosis in plants when exposed to metals. The role of copper efflux systems and the impact of copper on heme and chlorophyll biosynthesis in phototrophs are addressed.

Methotrexate enhances 5-aminolevulinic acid-mediated photodynamic therapy-induced killing of human SCC4 cells by upregulation of coproporphyrinogen oxidase.

BACKGROUND/PURPOSE: Topical 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) is effective for treatment of oral precancerous and cancerous lesions. This in vitro study tried to examine whether the SCC4 cell killing by ALA-PDT was enhanced by pretreatment of methotrexate (MTX). METHODS: To measure the SCC4 cell killing abilities by MTX-pretreated ALA-PDT (MTX-ALA-PDT), the SCC4 cells were pretreated with 0 mg/L, 0.001 mg/L, 0.01 mg/L, 0.1 mg/L, or 1 mg/L of MTX for 72 hours, then incubated with 0 mM, 0.0625 mM, 0.125 mM, 0.187 mM, 0.25 mM, or 0.375 mM ALA for 4 hours, and subsequently illuminated with a 640-nm light-emitting diode array at a light dose of 10 J/cm(2). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was conducted at 24 hours to quantify SCC4 cell survival rates after MTX-ALA-PDT treatment. Western blot analyses were used to examine the MTX-mediated enhancement in the expressions of the heme production-related enzymes, coproporphyrinogen oxidase (CPOX), protoporphyrinogen oxidase (PPOX), and ferrochelatase, in the MTX-preconditioned SCC4 cells. RESULTS: Pretreatment of SCC4 cells by 0.001 mg/L MTX for 72 hours resulted in a significant augmentation in MTX-ALA-PDT-induced killing of SCC4 cells (p < 0.05). The SCC4 cells treated with 0.001 mg/L MTX for 72 hours showed a significant and 1.65-fold increase in CPOX expression compared with the control SCC4 cells without MTX treatment (p < 0.05). However, no significant changes in the expressions of PPOX and ferrochelatase were observed in the SCC4 cells pretreated with different concentrations of MTX. CONCLUSION: MTX enhances ALA-PDT-induced SCC4 cell killing through upregulation of CPOX expression and subsequent increase in intracellular protoporphyrin IX production in SCC4 cells.

Mechanism of differentiation-enhanced photodynamic therapy for cancer: upregulation of coproporphyrinogen oxidase by C/EBP transcription factors.

The efficacy of photodynamic therapy (PDT) for epithelial cancers is increased when PDT is combined with calcitriol (Vit D), a form of differentiation therapy (DT). Here, we describe an underlying mechanism for this effect. Differentiation-promoting agents are known to upregulate CCAAT/enhancer-binding proteins (C/EBP), powerful regulators of cellular differentiation. In subcutaneous A431 tumors in mice, pretreatment with Vit D induced the expression of C/EBPß isoforms, and of coproporphyrinogen oxidase (CPO), a heme pathway enzyme responsible for the conversion of 5-aminolevulinic acid (ALA) into protoporphyrin IX (PpIX), the principal light-absorbing molecule during PDT. To further investigate this apparent link between C/EBPs and CPO, two cell lines (MEL and LNCaP) were exposed to differentiating agents, and levels of PpIX, C/EBPs, and CPO were measured. Differentiating agents, or transfection of C/EBP expression vectors, increased C/EBP and CPO levels in parallel. Focusing on approximately 1,300 bp of upstream CPO gene promoter, we tested the ability of recombinant C/EBPα, C/EBPß, C/EBPδ, and C/EBPζ to bind to CPO gene sequences [electrophoretic mobility shift assay (EMSA) assays] and to affect transcriptional activity (luciferase assays). Multiple C/EBP consensus binding sites were identified (15 for mouse, 18 for human). Individual probes representing each site bound to C/EBPs with characteristic affinities (strong, moderate, or weak), but when sites were inactivated in the context of the native promoter, transcriptional activity was reduced nearly equally for strong or weak sites. Cooperative interactions between regularly spaced C/EBP sites seem critical for CPO transcriptional regulation by differentiation therapy. These results provide a mechanistic rationale for DT/PDT combination therapy for cancer.

Homologous NF-YC2 subunit from Arabidopsis and tobacco is activated by photooxidative stress and induces flowering.

The transcription factor NF-Y consists of the three subunits A, B and C, which are encoded in Arabidopsis in large gene families. The multiplicity of the genes implies that NF-Y may act in diverse combinations of each subunit for the transcriptional control. We aimed to assign a function in stress response and plant development to NF-YC subunits by analyzing the expression of NF-Y genes and exploitation of nf-y mutants. Among the subunit family, NF-YC2 showed the strongest inducibility towards oxidative stress, e.g. photodynamic, light, oxidative, heat and drought stress. A tobacco NF-YC homologous gene was found to be inducible by photooxidative stress generated by an accumulation of the tetrapyrrole metabolite, coproporphyrin. Despite the stress induction, an Arabidopsis nf-yc2 mutant and NF-YC2 overexpressors did not show phenotypical differences compared to wild-type seedlings in response to photooxidative stress. This can be explained by the compensatory potential of other members of the NF-YC family. However, NF-YC2 overexpression leads to an early flowering phenotype that is correlated with increased FLOWERING LOCUS T-transcript levels. It is proposed that NF-YC2 functions in floral induction and is a candidate gene among the NF-Y family for the transcriptional activation upon oxidative stress.

Enhanced expression of coproporphyrinogen oxidase in malignant brain tumors: CPOX expression and 5-ALA-induced fluorescence.

In photodynamic diagnosis, 5-aminolevulinic acid (5-ALA) is widely used for the fluorescence-guided resection of malignant brain tumors, where 5-ALA is converted to protoporphyrin IX, which exhibits strong fluorescence. Little is known, however, about the detailed molecular mechanisms underlying 5-ALA-induced fluorescence. To resolve this issue, we analyzed transcriptome profiles for the genes encoding enzymes, transporters, and a transcription factor involved in the porphyrin-biosynthesis pathway. By quantitative real-time (qRT)-PCR, we measured the mRNA levels of those genes in a total of 20 tumor samples that had been surgically resected from brain tumor patients at the Department of Neurosurgery of Osaka Medical College from 2008 to 2009. We selected 10 tumor samples with no 5-ALA-induced fluorescence, among which 2 were glioblastomas and 8 were metastatic brain tumors. Another 10 tumor samples were selected with strong fluorescence, among which 7 were glioblastomas and 3 were metastatic brain tumors. The qRT-PCR analysis study of these latter 10 samples revealed predominantly high levels of the mRNA of the coproporphyrinogen oxidase (CPOX) gene. The high mRNA level of CPOX expression was significantly well correlated with the phenotype of strong 5-ALA-induced fluorescence (P = .0003). These findings were further confirmed by immunohistochemical studies with a CPOX-specific antibody. It is concluded that induction of CPOX gene expression is one of the key molecular mechanisms underlying the 5-ALA-induced fluorescence of malignant brain tumors. The induction mechanism for the CPOX gene in brain tumors remains to be elucidated.

Photodynamic therapy with hexenyl ester of 5-aminolevulinic acid induces necrotic cell death in salivary gland adenocarcinoma cells.

Photodynamic therapy has been developed as an alternative therapy of cancer. The aim of this study was to examine whether PDT with hexenyl ester of 5-aminolaevulinic acid (ALA-hx) inhibits the proliferation of the salivary gland adenocarcinoma SGT cells. Cell proliferation was examined by MTT assay. The gene expression of Coproporphyrinogen oxidase (CPO) and ROS production was also examined. Flow cytometry and in vivo Chorioallantoic membrane (CAM) assay was performed. ALA-hx PDT inhibited effectively the proliferation of SGT cells. Treatment of ALA-hx induced CPO mRNA expression and ROS was produced by ALA-hx PDT in SGT cells. Flow cytometry and LDH assay showed that ALA-hx PDT induced necrotic cell death rather than apoptosis in SGT cells. In vivo CAM assay showed that ALA-hx PDT induced tumor destruction by inducing necrosis. These results indicate that ALA-hx PDT effectively inhibits the proliferation of SGT cells by inducing necrosis.

Elucidation of the mechanism of mitochondrial iron loading in Friedreich's ataxia by analysis of a mouse mutant.

We used the muscle creatine kinase (MCK) conditional frataxin knockout mouse to elucidate how frataxin deficiency alters iron metabolism. This is of significance because frataxin deficiency leads to Friedreich's ataxia, a disease marked by neurologic and cardiologic degeneration. Using cardiac tissues, we demonstrate that frataxin deficiency leads to down-regulation of key molecules involved in 3 mitochondrial utilization pathways: iron-sulfur cluster (ISC) synthesis (iron-sulfur cluster scaffold protein1/2 and the cysteine desulferase Nfs1), mitochondrial iron storage (mitochondrial ferritin), and heme synthesis (5-aminolevulinate dehydratase, coproporphyrinogen oxidase, hydroxymethylbilane synthase, uroporphyrinogen III synthase, and ferrochelatase). This marked decrease in mitochondrial iron utilization and resultant reduced release of heme and ISC from the mitochondrion could contribute to the excessive mitochondrial iron observed. This effect is compounded by increased iron availability for mitochondrial uptake through (i) transferrin receptor1 up-regulation, increasing iron uptake from transferrin; (ii) decreased ferroportin1 expression, limiting iron export; (iii) increased expression of the heme catabolism enzyme heme oxygenase1 and down-regulation of ferritin-H and -L, both likely leading to increased "free iron" for mitochondrial uptake; and (iv) increased expression of the mammalian exocyst protein Sec15l1 and the mitochondrial iron importer mitoferrin-2 (Mfrn2), which facilitate cellular iron uptake and mitochondrial iron influx, respectively. Our results enable the construction of a model explaining the cytosolic iron deficiency and mitochondrial iron loading in the absence of frataxin, which is important for understanding the pathogenesis of Friedreich's ataxia.

Low-dose methotrexate enhances aminolevulinate-based photodynamic therapy in skin carcinoma cells in vitro and in vivo.

PURPOSE: To improve treatment efficacy and tumor cell selectivity of delta-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) via pretreatment of cells and tumors with methotrexate to enhance intracellular photosensitizer levels. EXPERIMENTAL DESIGN: Skin carcinoma cells, in vitro and in vivo, served as the model system. Cultured human SCC13 and HEK1 cells, normal keratinocytes, and in vivo skin tumor models were preconditioned with methotrexate for 72 h and then incubated with ALA for 4 h. Changes in protoporphyrin IX (PpIX) levels and cell survival after light exposure were assessed. RESULTS: Methotrexate preconditioning of monolayer cultures preferentially increased intracellular PpIX levels 2- to 4-fold in carcinoma cells versus normal keratinocytes. Photodynamic killing was synergistically enhanced by the combined therapy compared with PDT alone. Methotrexate enhancement of PpIX levels was achieved over a broad methotrexate concentration range (0.0003-1.0 mg/L; 0.6 nmol/L-2 mmol/L). PpIX enhancement correlated with changes in protein expression of key porphyrin pathway enzymes, approximately 4-fold increase in coproporphyrinogen oxidase and stable or slightly decreased expression of ferrochelatase. Differentiation markers (E-cadherin, involucrin, and filaggrin) were also selectively induced by methotrexate in carcinoma cells. In vivo relevance was established by showing that methotrexate preconditioning enhances PpIX accumulation in three models: (a) organotypic cultures of immortalized keratinocytes, (b) chemically induced skin tumors in mice; and (c) human A431 squamous cell tumors implanted subcutaneously in mice. CONCLUSION: Combination therapy using short-term exposure to low-dose methotrexate followed by ALA-PDT should be further investigated as a new combination modality to enhance efficacy and selectivity of PDT for epithelial carcinomas.

The Radical SAM Superfamily.

The radical S-adenosylmethionine (SAM) superfamily currently comprises more than 2800 proteins with the amino acid sequence motif CxxxCxxC unaccompanied by a fourth conserved cysteine. The charcteristic three-cysteine motif nucleates a [4Fe-4S] cluster, which binds SAM as a ligand to the unique Fe not ligated to a cysteine residue. The members participate in more than 40 distinct biochemical transformations, and most members have not been biochemically characterized. A handful of the members of this superfamily have been purified and at least partially characterized. Significant mechanistic and structural information is available for lysine 2,3-aminomutase, pyruvate formate-lyase, coproporphyrinogen III oxidase, and MoaA required for molybdopterin biosynthesis. Biochemical information is available for spore photoproduct lyase, anaerobic ribonucleotide reductase activation subunit, lipoyl synthase, and MiaB involved in methylthiolation of isopentenyladenine-37 in tRNA. The radical SAM enzymes biochemically characterized to date have in common the cleavage of the [4Fe-4S](1 +) -SAM complex to [4Fe-4S](2 +)-Met and the 5' -deoxyadenosyl radical, which abstracts a hydrogen atom from the substrate to initiate a radical mechanism.

S-adenosylmethionine as an oxidant: the radical SAM superfamily.

A recently discovered superfamily of enzymes function using chemically novel mechanisms, in which S-adenosylmethionine (SAM) serves as an oxidizing agent in DNA repair and the biosynthesis of vitamins, coenzymes and antibiotics. Members of this superfamily, the radical SAM enzymes, are related by the cysteine motif CxxxCxxC, which nucleates the [4Fe-4S] cluster found in each. A common thread in the novel chemistry of these proteins is the use of a strong reducing agent--a low-potential [4Fe-4S](1+) cluster--to generate a powerful oxidizing agent, the 5'-deoxyadenosyl radical, from SAM. Recent results are beginning to determine the unique biochemistry for some of the radical SAM enzymes, for example, lysine 2,3 aminomutase, pyruvate formate lyase activase and biotin synthase.

Methotrexate used in combination with aminolaevulinic acid for photodynamic killing of prostate cancer cells.

Photodynamic therapy (PDT) using 5-aminolaevulinic acid (ALA) to drive production of an intracellular photosensitiser, protoporphyrin IX (PpIX), is a promising cancer treatment. However, ALA-PDT is still suboptimal for thick or refractory tumours. Searching for new approaches, we tested a known inducer of cellular differentiation, methotrexate (MTX), in combination with ALA-PDT in LNCaP cells. Methotrexate alone promoted growth arrest, differentiation, and apoptosis. Methotrexate pretreatment (1 mg l(-1), 72 h) followed by ALA (0.3 mM, 4 h) resulted in a three-fold increase in intracellular PpIX, by biochemical and confocal analyses. After exposure to 512 nm light, killing was significantly enhanced in MTX-preconditioned cells. The reverse order of treatments, ALA-PDT followed by MTX, yielded no enhancement. Methotrexate caused a similar relative increase in PpIX, whether cells were incubated with ALA, methyl-ALA, or hexyl-ALA, arguing against a major effect upon ALA transport. Searching for an effect among porphyrin synthetic enzymes, we found that coproporphyrinogen oxidase (CPO) was increased three-fold by MTX at the mRNA and protein levels. Transfection of LNCaP cells with a CPO-expressing vector stimulated the accumulation of PpIX. Our data suggest that MTX, when used to modulate intracellular production of endogenous PpIX, may provide a new combination PDT approach for certain cancers.