DOPA pheomelanin is increased in nigral neuromelanin of Parkinson's disease.

Neuromelanin (NM) in dopaminergic neurons of human substantia nigra (SN) has a melanic component that consists of pheomelanin and eumelanin moieties and has been proposed as a key factor contributing to dopaminergic neuron vulnerability in Parkinson's disease (PD). While eumelanin is considered as an antioxidant, pheomelanin and related oxidative stress are associated with compromised drug and metal ion binding and melanoma risk. Using postmortem SN from patients with PD or Alzheimer's disease (AD) and unaffected controls, we identified increased L-3,4-dihydroxyphenylalanine (DOPA) pheomelanin and increased ratios of dopamine (DA) pheomelanin markers to DA in PD SN compared to controls. Eumelanins derived from both DOPA and DA were reduced in PD group. In addition, we report an increase in DOPA pheomelanin relative to DA pheomelanin in PD SN. In AD SN, we observed unaltered melanin markers despite reduced DOPA compared to controls. Furthermore, synthetic DOPA pheomelanin induced neuronal cell death in vitro while synthetic DOPA eumelanin showed no significant effect on cell viability. Our findings provide insights into the different roles of pheomelanin and eumelanin in PD pathophysiology. We anticipate our study will lead to further investigations on pheomelanin and eumelanin individually as biomarkers and possibly therapeutic targets for PD.

Pyruvate and lactate based hydrogel film inhibits UV radiation-induced skin inflammation and oxidative stress.

Solar skin damage is one of the most common diseases among outdoor workers. An important cause for the damage is the ultraviolet and infrared rays in sunlight, which are absorbed by the skin in large amounts, leading to severe skin inflammation and oxidative stress. Therefore, physical prevention by shielding the light from harmful wavelengths can be an effective method of skin protection from radiation. However, for existing skin lesions, prompt treatment is essential to avoid the aggravation of the injury and promote repair. Therefore, to improve the therapeutic effect on sun-damaged skin, we attempted to design a system with a dual purpose of eliminating toxic free radicals and modulating tissue inflammatory response. Here, we designed and synthesized a poly-acryloyl lysine (P-Ac-Lys) and polyvinyl alcohol-dihydroxyphenylalanine (PVA-DOPA) composite hydrogel (PAL@PVA-DOPA Hydrogel) loaded with lactate and pyruvate, that exhibites a good free radical scavenging activity and an excellent ability to modulate the inflammatory response. Experimental results showe that this hydrogel film could effectively reduce the UV-induced skin inflammation response, alleviate pathological damage and promote the recovery of the damaged skin.

Semi-Biosynthetic Production of Surface-Binding Adhesive Antimicrobial Peptides Using Intein-Mediated Protein Ligation.

Microbial infections remain a global health concern, calling for the urgent need to implement effective prevention measures. Antimicrobial peptides (AMPs) have been extensively studied as potential antimicrobial coating agents. However, an efficient and economical method for AMP production is lacking. Here, we synthesized the direct coating adhesive AMP, NKC-DOPA5, composed of NKC, a potent AMP, and repeats of the adhesive amino acid 3,4-dihydroxyphenylalanine (DOPA) via an intein-mediated protein ligation strategy. NKC was expressed as a soluble fusion protein His-NKC-GyrA (HNG) in Escherichia coli, comprising an N-terminal 6× His-tag and a C-terminal Mxe GyrA intein. The HNG protein was efficiently produced in a 500-L fermenter, with a titer of 1.63 g/L. The NKC-thioester was released from the purified HNG fusion protein by thiol attack and subsequently ligated with chemically synthesized Cys-DOPA5. The ligated peptide His-NKC-Cys-DOPA5 was obtained at a yield of 88.7%. The purified His-NKC-Cys-DOPA5 possessed surface-binding and antimicrobial properties identical to those of the peptide obtained via solid-phase peptide synthesis. His-NKC-Cys-DOPA5 can be applied as a practical and functional antimicrobial coating to various materials, such as medical devices and home appliances.

Biosynthesis and Genetic Incorporation of 3,4-Dihydroxy-L-Phenylalanine into Proteins in Escherichia coli.

While 20 canonical amino acids are used by most organisms for protein synthesis, the creation of cells that can use noncanonical amino acids (ncAAs) as additional protein building blocks holds great promise for preparing novel medicines and for studying complex questions in biological systems. However, only a small number of biosynthetic pathways for ncAAs have been reported to date, greatly restricting our ability to generate cells with ncAA building blocks. In this study, we report the creation of a completely autonomous bacterium that utilizes 3,4-dihydroxy-L-phenylalanine (DOPA) as its 21st amino acid building block. Like canonical amino acids, DOPA can be biosynthesized without exogenous addition and can be genetically incorporated into proteins in a site-specific manner. Equally important, the protein production yields of DOPA-containing proteins from these autonomous cells are greater than those from cells exogenously fed with 9 mM DOPA. The unique catechol moiety of DOPA can be used as a versatile handle for site-specific protein functionalizations via either oxidative coupling or strain-promoted oxidation-controlled cyclooctyne-1,2-quinone (SPOCQ) cycloaddition reactions. We further demonstrate the use of these autonomous cells in preparing fluorophore-labeled anti-human epidermal growth factor 2 (HER2) antibodies for the detection of HER2 expression on cancer cells.

Melanocyte progenitor cells reside in human subcutaneous adipose tissue.

Based on the assumption that some progenitor cells in an organ might reside in neighboring adipose tissue, we investigated whether melanocyte progenitor cells reside in human subcutaneous adipose tissue. First, we examined the expression of human melanoma black 45 (HMB45) and microphthalmia-associated transcription factor (MITF) in undifferentiated adipose-derived stem cells (ADSCs) by immunostaining, RT-PCR, and western blotting. These two markers were detected in undifferentiated ADSCs, and their expression levels were increased in differentiated ADSCs in melanocyte-specific culture medium. Other melanocytic markers (Melan A, MATP, Mel2, Mel EM, tyrosinase, KIT, and PAX3) were also detected at variable levels in undifferentiated ADSCs, and the expression of some markers was increased during differentiation into the melanocyte lineage. We further showed that ADSCs differentiated in melanocyte-specific culture medium localized in the basal layer and expressed tyrosinase and HMB45 in a 3D epidermal culture system. Melanin deposits were also induced by ultraviolet-light-B (UVB) irradiation. These results demonstrate that melanocyte progenitor cells reside in human subcutaneous adipose tissue and that these cells might have the potential to differentiate into mature melanocytes. Melanocyte and keratinocyte progenitors residing in human subcutaneous tissue can be used for the treatment of skin diseases and skin rejuvenation in the future.

Candida glabrata produces a melanin-like pigment that protects against stress conditions encountered during parasitism.

Aim: Melanin has been linked to pathogenesis in several fungi. They often produce melanin-like pigments in the presence of L-dihydroxyphenylalanine (L-DOPA), but this is poorly studied in Candida glabrata. Methods & materials:C. glabrata was grown in minimal medium with or without L-DOPA supplementation and submitted to a chemical treatment with denaturant and hot acid. Results:C. glabrata turned black when grown in the presence of L-DOPA, whereas cells grown without L-DOPA supplementation remained white. Biophysical properties demonstrated that the pigment was melanin. Melanized C. glabrata cells were effectively protected from azoles and amphotericin B, incubation at 42°C and macrophage killing. Conclusion: In the presence of L-DOPA, C. glabrata produces melanin, increases antifungal resistance and enhances host survival.

Aim: Melanin is a pigment that can help fungi to cause disease. Fungi often produce melanin-like pigments in the presence of L-dihydroxyphenylalanine (L-DOPA), but this is poorly studied in Candida glabrata, a yeast species that can cause human disease. Methods & materials:C. glabrata was grown in minimal medium with or without L-DOPA supplementation and submitted to a chemical treatment to isolate melanin. Results:C. glabrata turned black when grown in the presence of L-DOPA, whereas cells grown without L-DOPA supplementation remained white. Several experiments demonstrated that the black pigment was melanin. Melanized C. glabrata cells were effectively protected from antifungal drugs, incubation at 42°C and killing by cells of the immune system. Conclusion: In the presence of L-DOPA, C. glabrata produces melanin, increases antifungal resistance and has enhanced survival in contact with immunologic defense cells.

High 18F-FDOPA PET Uptake in Primary Central Nervous System Lymphoma.

We present a 42-year-old woman with primary central nervous system lymphoma (PCNSL) and strong F-FDOPA PET uptake. F-FDOPA PET has high diagnostic accuracy in gliomas and brain metastases. The L-type amino acid transporter 1, targeted by F-FDOPA and C-MET PET, is a cell-type transporter usually upregulated in malignant tumors, including PCNSL. In this line, strong uptake was already shown with C-MET in PCNSL. We report the same findings with F-FDOPA. Consequently, PCNSL is a possible differential neoplastic diagnosis of F-FDOPA uptake among neoplastic lesions.

Maximum Uptake and Hypermetabolic Volume of 18F-FDOPA PET Estimate Molecular Status and Overall Survival in Low-Grade Gliomas: A PET and MRI Study.

PURPOSE: We evaluated F-FDOPA PET and MRI characteristics in association with the molecular status and overall survival (OS) in a large number of low-grade gliomas (LGGs). METHODS: Eighty-six patients who underwent F-FDOPA PET and MRI and were diagnosed with new or recurrent LGGs were retrospectively evaluated with respect to their isocitrate dehydrogenase (IDH) and 1p19q status (10 IDH wild type, 57 mutant, 19 unknown; 1p19q status in IDH mutant: 20 noncodeleted, 37 codeleted). After segmentation of the hyperintense area on fluid-attenuated inversion recovery image (FLAIRROI), the following were calculated: normalized SUVmax (nSUVmax) of F-FDOPA relative to the striatum, F-FDOPA hypermetabolic volume (tumor-to-striatum ratios >1), FLAIRROI volume, relative cerebral blood volume, and apparent diffusion coefficient within FLAIRROI. Receiver operating characteristic curve and Cox regression analyses were performed. RESULTS: PET and MRI metrics combined with age predicted the IDH mutation and 1p19q codeletion statuses with sensitivities of 73% and 76% and specificities of 100% and 94%, respectively. Significant correlations were found between OS and the IDH mutation status (hazard ratio [HR] = 4.939), nSUVmax (HR = 2.827), F-FDOPA hypermetabolic volume (HR = 1.048), and FLAIRROI volume (HR = 1.006). The nSUVmax (HR = 151.6) for newly diagnosed LGGs and the F-FDOPA hypermetabolic volume (HR = 1.038) for recurrent LGGs demonstrated significant association with OS. CONCLUSIONS: Combining F-FDOPA PET and MRI with age proved useful for predicting the molecular status in patients with LGGs, whereas the nSUVmax and F-FDOPA hypermetabolic volume may be useful for prognostication.

Effects of anti-wrinkle and skin-whitening fermented black ginseng on human subjects and underlying mechanism of action.

The aim of this study was to determine the effects of anti-wrinkle and skin-whitening of fermented black ginseng (FBG) in human subjects and to examine underlying biochemical mechanisms of action. A clinical study was performed to evaluate efficacy and safety using a 1% FBG cream formulation. Twenty-three subjects were recruited and instructed to apply control or FBG creams each on half of their face twice daily for 8 weeks. After 8 weeks FBG cream significantly reduced appearance of eye wrinkles compared to prior to exposure and control cream. Skin color was significantly brightened using FBG cream in comparison with control cream. To determine the mechanism of actions involved in anti-wrinkle and skin-whitening effects various concentrations of FBG were applied to human fibroblast CCD-986sk and mouse melanoma B16F1 cells. Collagen synthesis in CCD-986sk cells was improved significantly at 1, 3, 10, or 30 µg/ml of FBG. At 30 µg/ml, FBG significantly inhibited (73%) collagenase, and matrix metalloproteinase-1 (MMP-1) compared to control. Tyrosinase activity and DOPA (3,4-dihydroxy-L-phenylalanine) oxidation were significantly decreased at all tested concentrations. Melanin production in B16F1 cells was concentration-dependently reduced 15% to 60% by all concentrations of FBG. These results suggested that a 1% FBG cream exerted anti-wrinkle and skin-whitening effects.

Compartmentalized processing of catechols during mussel byssus fabrication determines the destiny of DOPA.

Inspired largely by the role of the posttranslationally modified amino acid dopa (DOPA) in mussel adhesion, catechol functional groups have become commonplace in medical adhesives, tissue scaffolds, and advanced smart polymers. Yet, the complex redox chemistry of catechol groups complicates cross-link regulation, hampering fabrication and the long-term stability/performance of mussel-inspired polymers. Here, we investigated the various fates of DOPA residues in proteins comprising mussel byssus fibers before, during, and after protein secretion. Utilizing a combination of histological staining and confocal Raman spectroscopy on native tissues, as well as peptide-based cross-linking studies, we have identified at least two distinct DOPA-based cross-linking pathways during byssus fabrication, achieved by oxidative covalent cross-linking or formation of metal coordination interactions under reducing conditions, respectively. We suggest that these end states are spatiotemporally regulated by the microenvironments in which the proteins are stored prior to secretion, which are retained after formation-in particular, due to the presence of reducing moieties. These findings provide physicochemical pathways toward greater control over properties of synthetic catechol-based polymers and adhesives.

Quantitative changes in the secretion of exosomes from keratinocytes homeostatically regulate skin pigmentation in a paracrine manner.

The content and distribution of melanin in the epidermis determines the wide variety of skin colors associated with ethnic/racial diversity. Although it was previously reported that qualitative changes in keratinocyte-derived exosomes regulate melanocyte pigmentation in vitro, their practical involvement, especially in skin color development in vivo, has remained unclear. To address this unexplained scientific concern, the correlation of epidermal exosomes isolated from human skin tissues with melanosomal protein expression levels was demonstrated in this study for the first time. After confirming the quantitative effect of human keratinocyte-derived exosomes on human melanocyte activation, even in the absence of ultraviolet B (UV-B) exposure, the impact of exosomes secreted from UV-B-irradiated keratinocytes on melanogenesis was consistently detected, which suggests their constitutive role in regulating cutaneous pigmentation. Additionally, both a specific exosome secretion inducer and a suppressor were consistently found to significantly control melanin synthesis in a co-culture system composed of keratinocytes and melanocytes as well as in an ex vivo skin culture system. These results suggest that quantitative changes, in addition to already known qualitative changes, in exosomes secreted from human epidermal keratinocytes homeostatically regulate melanogenic activity in a paracrine manner, which leads to skin color determination.

Uptake of Pineal Gland Visible on 18F-DOPA PET/CT With Last Generation of SiPM PET/CT.

A 17-year-old girl underwent an F-DOPA brain PET/CT on a new high-resolution digital SiPM PET/CT (Vision600 Siemens) to explore a suspicion of recurrence of a pilocytic astrocytoma. This study showed a local recurrence, but a second intense focal uptake was visible above, more intense than striata. On fused MRI, this was the pineal gland considered as physiological. This physiological uptake, due to a pineal DOPA decarboxylase activity, has also been observed with this PET system in other patients with F-DOPA to explore movement disorders. New high-resolution PET can show uptake of small structures by being less dependent to partial volume effect.

Matrix-assisted pulsed laser evaporation of ß-glucosidase from a dopa/quinone target.

ß-glucosidase (BG) plays a key role in determining the efficiency of the enzymatic complex cellulase for the degradation of cellulose into sugars. It hydrolyses the cellobiose, an inhibitor of the enzymatic complex. Therefore, the immobilization of BG is a great challenge for the industrial application of cellulases. Cellulases usually contains a BG amount insufficient to avoid inhibition by cellobiose. Here the BG was immobilized by matrix assisted pulsed laser evaporation (MAPLE) technique. The frozen matrix was composed of water, water/m-DOPA and water/m-DOPA/quinone. The effect of the excipients on the final conformation of the enzyme after the MAPLE processing was determined. The enzyme secondary structure was studied by FTIR analysis. The catalytic performances of the deposited films were tested in the cellobiose hydrolysis reaction. The results demonstrate that the presence of the oxidized form of m-DOPA, the O-quinone form, can protect the protein native structure, with the laser inducing little or no damage. In fact, only the samples deposited from this target preserved the secondary structure of the polypeptide chain and allowed a complete hydrolysis of cellobiose for four consecutive runs, showing a high operational stability of the biocatalyst.

Serum 5-S-cysteinyl-dopa levels as a predictive marker for the efficacy of nivolumab in advanced malignant melanoma.

PURPOSE: With the recent developments in novel molecular targeted therapy such as immune-checkpoint blockades, serine/threonine-protein kinase B-Raf, and mitogen-activated protein kinase kinase inhibitors, the prognosis of advanced malignant melanoma has been improving. 5-S-cysteinyl-dopa (5-S-CD), a precursor of pheomelanin, has been previously revealed to be a useful biomarker for advanced-stage malignant melanoma, especially in patients with distant metastases. Here, we aimed to assess and compare the utility of serum 5-S-CD and lactate dehydrogenase levels as markers for predicting the effects of nivolumab in advanced malignant melanoma. METHODS: Baseline serum 5-S-CD and lactate dehydrogenase levels in patients with unresectable stage IIIC and IV malignant melanoma treated with nivolumab (n = 21) were analyzed to determine their utility as predictive markers for survival. We also analyzed the prognostic value of these markers among patients with only stage IV malignant melanoma (n = 17). RESULTS: Our analysis showed that patients with baseline serum 5-S-CD levels >25.0 nmol/L had significantly poor prognosis. In contrast, serum lactate dehydrogenase levels at the upper limit of the normal range did not exhibit such changes. CONCLUSIONS: Serum 5-S-CD levels have the potential to be an excellent predictive marker for the efficacy of nivolumab therapy in patients with advanced malignant melanoma.

Protective Effect of Botulinum Toxin against Ultraviolet-Induced Skin Pigmentation.

BACKGROUND: Hyperpigmentation following ultraviolet irradiation has cosmetic concerns. Botulinum toxin type A can favorably affect skin pigmentation. However, the mechanism of skin pigmentation is unclear. METHODS: In vitro, human epidermal melanocytes were co-cultured with human keratinocytes. After cells were treated with botulinum toxin type A, cell morphology, proliferation, and dendricity were analyzed, and immunofluorescence, tyrosinase activity, and melanin contents were determined. To evaluate the effect of botulinum toxin type A on ultraviolet B-irradiated mouse skin, ultraviolet B alone was applied to one side of the back of each mouse as a control, whereas ultraviolet B plus injection of botulinum toxin type A was applied to the contralateral side. Skin pigmentation, histology, and the number of dihydroxyphenylalanine-positive melanocytes were evaluated. The L* colorimeter value was measured. Enzyme-linked immunosorbent assay determinations of basic fibroblast growth factor, interleukin-1 alpha, and prostaglandin E2 were performed. RESULTS: Immunohistochemical staining revealed botulinum toxin type A in the cytoplasm of melanocytes and in the positive control. In vitro, melanocyte dendricity and melanin contents were decreased slightly but significantly (p < 0.05) after botulinum toxin type A treatment. In vivo, botulinum toxin type A suppressed skin pigmentation. The number of dihydroxyphenylalanine-positive melanocytes was also significantly lower than in the control side. Tyrosinase activity and melanin content were also significantly reduced (p < 0.05). Botulinum toxin type A also significantly reduced the amounts of basic fibroblast growth factor, interleukin-1 alpha, and prostaglandin E2 (all p < 0.05). CONCLUSION: Botulinum toxin type A can suppress epidermal melanogenesis through both direct and indirect mechanisms.

Effect of fatty acids on melanogenesis and tumor cell growth in melanoma cells.

Fatty acids have various physiological effects on melanoma. For example, palmitic acid (PA) increases melanin levels; linoleic acid and DHA decrease melanin levels; and DHA suppresses tumor growth. In this study, we focused on the relationship between the structure of fatty acids and their physiological effects in melanoma to examine the likely mechanisms of action. We showed that saturated fatty acids and PUFAs display opposing effects on melanin content in melanoma cells. Likewise, PA and EPA have opposing effects in terms of actin polymerization. Our findings suggest that PA and EPA change melanin content in melanoma to alter melanosome trafficking by modulating actin polymerization. Here, we also examined the mechanism of the anti-tumor effect of DHA. We found that DHA interacts with receptor for activated C kinase 1 and represses melanoma cell proliferation by suppressing protein kinase C signaling. Our results suggest a new mechanism to explain the physiological effects of fatty acids.

Fast enzymatic synthesis of n.c.a. 6-[18 F]fluorodopamine (FDA) from n.c.a. 6-[18 F]FDOPA and the fate of 6-FDOPA and 6-FDA in neuroblastoma and Caki-1 cells after their uptake.

The catecholamine analogue [123 I]mIBG has been used for scintigraphic imaging of neuroblastoma since 1984. It is taken up by the noradrenaline transporter (NAT), which is present in most neuroblastoma cells. An alternative imaging method could be PET with 6-[18 F]fluorodopamine, which is also taken up by NAT, but-in contrast to mIBG-also by dopamine transporter (DAT), present in neuroblastoma cells (NAT > DAT). An enzymatic method was established allowing a rapid, quantitative transformation of FDOPA to FDA by DOPA decarboxylase within 25 minutes. This strategy was applied to [18 F]FDOPA, which was produced via nucleophilic synthesis (RCY 15%, 10 GBq, 50 GBq/µmol) and subsequently converted to [18 F]FDA (RCY 35%-50%, n = 5). Uptake and metabolism of FDOPA and FDA were analyzed in human Kelly and SK-N-SH neuroblastoma cell lines and in human Caki-1 kidney cells that can take up catecholamines and mIBG via an organic cation transporter (OCT). FDOPA and FDA were taken up by all three cells, but FDOPA could only be converted to FDA in neuroblastoma cells. As today, [18 F]FDOPA is well available in high yields, efficient enzymatic conversion to [18 F]FDA to be used for NAT/DAT PET imaging in neuroendocrine tumors is an attractive, alternative synthesis route.

18F-DOPA uptake does not correlate with IDH mutation status and 1p/19q co-deletion in glioma.

OBJECTIVE: The role of amino acid positron emission tomography (PET) in glioma grading and outcome prognostication has not yet been well established. This is particularly true in the context of the new WHO 2016 classification, which introduced a definition of glioma subtypes primarily based on molecular fingerprints. The aim of the present study was to correlate 3,4­dihydroxy­6­[18F]­fluoro-L­phenylalanine (F-DOPA) uptake parameters with IDH mutation, 1p/19q status, and survival outcomes in patients with glioma. METHODS: The study population consisted of 33 patients (17 M/16 F, mean age: 46 ± 13 years) who underwent F-DOPA PET/CT for the evaluation of tumor extent before the start of chemo or radiotherapy. The presence of IDH mutation and 1p/19q status was assessed in all the cases. Tumor volume and semiquantitative uptake parameters, namely SUVmax, tumor-to-normal brain ratio and tumor-to-normal striatum ratio, were calculated for each tumor. Imaging-derived parameters were compared between patients stratified according to molecular fingerprints, using parametric or non-parametric tests, where appropriate. The Kaplan-Meier method was used to assess differences of overall survival (OS) and progression-free survival (PFS) between groups. PET parameters were also tested as prognostic factors in univariate Cox survival regression models. RESULTS: There were 12 IDH-wild-type and 21 IDH-mutant patients. Stratification according to 1p/19q co-deletion resulted in 20 non-co-deleted and 13 co-deleted patients. Median follow-up time from PET/CT exam was 30.5 months (range 3.5-74 months). Semiquantitative uptake parameters did correlate neither with IDH mutation nor with 1p/19q status. Uptake was similar in low-grade and high-grade tumors, respectively. In addition, F-DOPA uptake parameters, macroscopic tumor volume, or tumor grade did not stratify OS, while a correlation between SUVmax and PFS was shown in the subgroup of astrocytomas. On the other hand, IDH mutation status and presence of 1p/19q co-deletion had a significant impact on survival outcomes. The prognostic value of IDH mutation status was also confirmed in the subgroup of patients with astrocytic tumors. CONCLUSIONS: F-DOPA uptake parameters do not correlate with tumor molecular and histological characteristics. The predictive value of PET-derived parameters on outcomes of survival is limited.

Polymerizing Like Mussels Do: Toward Synthetic Mussel Foot Proteins and Resistant Glues.

A novel strategy to generate adhesive protein analogues by enzyme-induced polymerization of peptides is reported. Peptide polymerization relies on tyrosinase oxidation of tyrosine residues to Dopaquinones, which rapidly form cysteinyldopa-moieties with free thiols from cysteine residues, thereby linking unimers and generating adhesive polymers. The resulting artificial protein analogues show strong adsorption to different surfaces, even resisting hypersaline conditions. Remarkable adhesion energies of up to 10.9 mJ m-2 are found in single adhesion events and average values are superior to those reported for mussel foot proteins that constitute the gluing interfaces.

Metal-free class Ie ribonucleotide reductase from pathogens initiates catalysis with a tyrosine-derived dihydroxyphenylalanine radical.

All cells obtain 2'-deoxyribonucleotides for DNA synthesis through the activity of a ribonucleotide reductase (RNR). The class I RNRs found in humans and pathogenic bacteria differ in (i) use of Fe(II), Mn(II), or both for activation of the dinuclear-metallocofactor subunit, ß; (ii) reaction of the reduced dimetal center with dioxygen or superoxide for this activation; (iii) requirement (or lack thereof) for a flavoprotein activase, NrdI, to provide the superoxide from O2; and (iv) use of either a stable tyrosyl radical or a high-valent dimetal cluster to initiate each turnover by oxidizing a cysteine residue in the α subunit to a radical (Cys•). The use of manganese by bacterial class I, subclass b-d RNRs, which contrasts with the exclusive use of iron by the eukaryotic Ia enzymes, appears to be a countermeasure of certain pathogens against iron deprivation imposed by their hosts. Here, we report a metal-free type of class I RNR (subclass e) from two human pathogens. The Cys• in its α subunit is generated by a stable, tyrosine-derived dihydroxyphenylalanine radical (DOPA•) in ß. The three-electron oxidation producing DOPA• occurs in Escherichia coli only if the ß is coexpressed with the NrdI activase encoded adjacently in the pathogen genome. The independence of this new RNR from transition metals, or the requirement for a single metal ion only transiently for activation, may afford the pathogens an even more potent countermeasure against transition metal-directed innate immunity.