Aziridine Formation by a FeII /α-Ketoglutarate Dependent Oxygenase and 2-Aminoisobutyrate Biosynthesis in Fungi.

Aziridine is a characteristically reactive molecule with increased bioactivity due to its strained ring structure. Here, we investigated the biosynthesis of 2-aminoisobutyric acid (AIB) in Penicillium, and successfully reconstituted the three-step biosynthesis from L-Val to AIB in vitro. This previously unknown aziridine formation pathway proceeded with the non-heme iron and α-ketoglutarate-dependent (FeII /αKG) oxygenase TqaL, followed by aziridine ring opening by the haloalkanoic acid dehalogenase (HAD)-type hydrolase TqaF, and subsequent oxidative decarboxylation by the NovR/CloR-like non-heme iron oxygenase TqaM. Furthermore, the X-ray crystal structure of the C-N bond forming FeII /αKG oxygenase TqaL was solved at 2.0 Šresolution. This work presents the first molecular basis for aziridine biogenesis, thereby expanding the catalytic repertoire of the FeII /αKG oxygenases. We also report the unique aziridine ring opening by a HAD-type hydrolase and the remarkable oxidative decarboxylation by a non-heme iron oxygenase to produce AIB.

Directed evolution of the B. subtilis nitroreductase YfkO improves activation of the PET-capable probe SN33623 and CB1954 prodrug.

OBJECTIVES: To use directed evolution to improve YfkO-mediated reduction of the 5-nitroimidazole PET-capable probe SN33623 without impairing conversion of the anti-cancer prodrug CB1954. RESULTS: Two iterations of error-prone PCR, purifying selection, and FACS sorting in a DNA damage quantifying GFP reporter strain were used to identify three YfkO variants able to sensitize E. coli host cells to at least 2.4-fold lower concentrations of SN33623 than the native enzyme. Two of these variants were able to be purified in a functional form, and in vitro assays revealed these were twofold and fourfold improved in kcat/KM with SN33623 over wild type YfkO. Serendipitously, the more-active variant was also nearly fourfold improved in kcat/KM versus wild type YfkO in converting CB1954 to a genotoxic drug. CONCLUSIONS: The enhanced activation of the PET imaging probe SN33623 and CB1954 prodrug exhibited by the lead evolved variant of YfkO offers prospects for improved enzyme-prodrug therapy.

Insight into enzyme-catalyzed aziridine formation mechanism in ficellomycin biosynthesis.

Ficellomycin is an aziridine-containing antibiotic, produced by Streptomyces ficellus. Based on the newly identified ficellomycin gene cluster and the assigned functions of its genes, a possible pathway for aziridine ring formation in ficellomycin was proposed, which is a complex process involving at least 3 enzymatic steps. To obtain support for the proposed mechanism, the targeted genes encoding sulfate adenylyltransferase, adenylsulfate kinase, and a putative sulfotransferase were respectively disrupted and the subsequent analysis of their fermentation products revealed that all the three genes were involved in aziridine formation. To further confirm the mechanism, the key gene encoding a putative sulfotransferase was over expressed in Escherichia coli Rosseta (DE3). Enzyme assays indicated that the expressed sulfotransferase could specifically transfer a sulfo group from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) onto the hydroxyl group of (R)-(-)-2-pyrrolidinemethanol. This introduces a good leaving group in the form of the sulfated hydroxyl moiety, which is then converted into an aziridine ring through an intramolecular nucleophilic attack by the adjacent secondary amine. The sulfation/intramolecular cyclization reaction sequence maybe a general strategy for aziridine biosynthesis in microorganisms. Discovery of this mechanism revealed an enzyme-catalyzed route for the synthesis of aziridine-containing reagents and provided an important insight into the functional diversity of sulfotransferases.

Hypoxia-Induced Pro-Protein Therapy Assisted by a Self-Catalyzed Nanozymogen.

The success of intracellular protein therapy demands efficient delivery and selective protein activity in diseased cells. Therefore, a cascaded nanozymogen consisting of a hypoxia-activatable pro-protein, a hypoxia-inducing protein, and a hypoxia-strengthened intracellular protein delivery nanovehicle was developed. RPAB, an enzymatically inactive pro-protein of RNase, reversibly caged with hypoxia-cleavable azobenzene, was delivered with glucose oxidase (GOx) using hypoxia-responsive nanocomplexes (NCs) consisting of azobenzene-cross-linked oligoethylenimine (AOEI) and hyaluronic acid (HA). Upon NC-mediated delivery into cancer cells, GOx catalyzed glucose decomposition and aggravated tumoral hypoxia, which drove the recovery of RPAB back to the hydrolytically active RNase and expedited the degradation of AOEI to release more protein cargoes. Thus, the catalytic reaction of the nanozymogen was self-accelerated and self-cycled, ultimately leading to a cooperative anti-cancer effect between GOx-mediated starvation therapy and RNase-mediated pro-apoptotic therapy.

Evaluating the abilities of diverse nitroaromatic prodrug metabolites to exit a model Gram negative vector for bacterial-directed enzyme-prodrug therapy.

Gene-directed enzyme-prodrug therapy (GDEPT) employs tumour-tropic vectors including viruses and bacteria to deliver a genetically-encoded prodrug-converting enzyme to the tumour environment, thereby sensitising the tumour to the prodrug. Nitroreductases, able to activate a range of promising nitroaromatic prodrugs to genotoxic metabolites, are of great interest for GDEPT. The bystander effect (cell-to-cell transfer of activated prodrug metabolites) has been quantified for some nitroaromatic prodrugs in mixed multilayer human cell cultures, however while these provide a good model for viral DEPT (VDEPT) they do not inform on the ability of these prodrug metabolites to exit bacterial vectors (relevant to bacterial-DEPT (BDEPT)). To investigate this we grew two Escherichia coli strains in co-culture; an activator strain expressing the nitroreductase E. coli NfsA and a recipient strain containing an SOS-GFP DNA damage responsive gene construct. In this system, induction of GFP by reduced prodrug metabolites can only occur following their transfer from the activator to the recipient cells. We used this to investigate five clinically relevant prodrugs: metronidazole, CB1954, nitro-CBI-DEI, and two dinitrobenzamide mustard prodrug analogues, PR-104A and SN27686. Consistent with the bystander efficiencies previously measured in human cell multilayers, reduced metronidazole exhibited little bacterial cell-to-cell transfer, whereas nitro-CBI-DEI was passed very efficiently from activator to recipient cells post-reduction. However, in contrast with observations in human cell multilayers, the nitrogen mustard prodrug metabolites were not effectively passed between the two bacterial strains, whereas reduced CB1954 was transferred efficiently. Using nitroreductase enzymes that exhibit different biases for the 2- versus 4-nitro substituents of CB1954, we further showed that the 2-nitro reduction products exhibit substantially higher levels of bacterial cell-to-cell transfer than the 4-nitro reduction products, consistent with their relative bystander efficiencies in human cell culture. Overall, our data suggest that prodrugs may differ in their suitability for VDEPT versus BDEPT applications and emphasise the importance of evaluating an enzyme-prodrug partnership in an appropriate context for the intended vector.

Distinct activation mechanisms trigger the trypanocidal activity of DNA damaging prodrugs.

Quinone-based compounds have been exploited to treat infectious diseases and cancer, with such chemicals often functioning as inhibitors of key metabolic pathways or as prodrugs. Here, we screened an aziridinyl 1,4-benzoquinone (ABQ) library against the causative agents of trypanosomiasis, and cutaneous leishmaniasis, identifying several potent structures that exhibited EC50 values of <100 nM. However, these compounds also displayed significant toxicity towards mammalian cells indicating that they are not suitable therapies for systemic infections. Using anti-T. brucei ABQs as chemical probes, we demonstrated that these exhibit different trypanocidal modes of action. Many functioned as type I nitroreductase (TbNTR) or cytochrome P450 reductase (TbCPR) dependent prodrugs that, following activation, generate metabolites which promote DNA damage, specifically interstrand crosslinks (ICLs). Trypanosomes lacking TbSNM1, a nuclease that specifically repairs ICLs, are hypersensitive to most ABQ prodrugs, a phenotype exacerbated in cells also engineered to express elevated levels of TbNTR or TbCPR. In contrast, ABQs that contain substituent groups on the biologically active aziridine do not function as TbNTR or TbCPR-activated prodrugs and do not promote DNA damage. By unravelling how ABQs mediate their activities, features that facilitate the desired anti-parasitic growth inhibitory effects could be incorporated into new, safer compounds targeting these neglected tropical diseases.

Non-natural carbenoid and nitrenoid insertion reactions catalyzed by heme proteins.

Despite increasing interest in using enzymes as tools for synthesis, many reactions discovered through the creativity of synthetic chemists remain beyond the scope of biocatalysis. This vacancy in the field has compelled researchers to develop strategies to adapt protein scaffolds for new reactivity. Heme proteins have recently been shown to activate synthetic precursors to generate reactive metallocarbenoid and metallonitrenoid species that enable the biosynthetic construction of novel C-C, C-N, and other bonds using mechanisms not previously explored by Nature. By interrogating heme proteins with synthetic, non-natural reagents, scientists are merging the reaction space traditionally dominated by organocatalysis and transition metal catalysis with the mild reaction conditions, selectivity, and adaptability imparted by native protein scaffolds.

Heterologous Overexpression and Biochemical Characterization of a Nitroreductase from Gluconobacter oxydans 621H.

A NADPH-dependent and FMN-containing nitroreductase (Gox0834) from Gluconobacter oxydans was cloned and heterogeneously expressed in Escherichia coli. The purified enzyme existed as a dimer with an apparent molecular mass of about 31.4 kDa. The enzyme displayed broad substrate specificity and reduced a variety of mononitrated, polynitrated, and polycyclic nitroaromatic compounds to the corresponding amino products. The highest activity was observed for the reduction of CB1954 (5-(1-aziridinyl)-2,4-dinitrobenzamide). The enzyme kinetics analysis showed that Gox0834 had relatively low K m (54 ± 11 µM) but high k cat/K m value (0.020 s(-1)/µM) for CB1954 when compared with known nitroreductases. Nitrobenzene and 2,4,6-trinitrotoluene (TNT) were preferred substrates for this enzyme with specific activity of 11.0 and 8.9 µmol/min/mg, respectively. Gox0834 exhibited a broad temperature optimum of 40-60 °C for the reduction of CB1954 with a pH optimum between 7.5 and 8.5. The purified enzyme was very stable below 37 °C over a broad pH range of 6.0-10.0. These characteristics suggest that the nitroreductase Gox0834 may be a possible candidate for catalyzing prodrug activation, bioremediation, or biocatalytic processes.

The Inhibition of Cysteine Proteases Rhodesain and TbCatB: A Valuable Approach to Treat Human African Trypanosomiasis.

Human African Trypanosomiasis (HAT) is an endemic parasitic disease of sub-Saharan Africa, caused by two subspecies of protozoa belonging to Trypanosoma genus: T. brucei gambiense and T. brucei rhodesiense. In this context the inhibition of the papain-family cysteine proteases rhodesain and TbCatB has to be considered a promising strategy for HAT treatment. Rhodesain, the major cathepsin L-like cysteine protease of T. brucei rhodesiense, is a lysosomal protease essential for parasite survival. It is involved in parasite invasivity, allowing it to cross the blood-brain barrier (BBB) of the human host, causing the second lethal stage of the disease. Moreover, it plays an important role in immunoevasion, being involved in the turnover of variant surface glycoproteins of the T. brucei coat and in the degradation of immunoglobulins, avoiding a specific immune response by the host cells. On the other hand TbCatB, a cathepsin B-like cysteine protease, present in minor abundance in T. brucei, showed a key role in the degradation of host transferrin, which is necessary for iron acquisition by the parasite. In this review article we now discuss the most active peptide, peptidomimetic and non-peptide rhodesain and TbCatB inhibitors as valuable strategy to treat HAT, due also to the complementary role of the two T. brucei proteases.

Study of Bioreductive Anticancer Agent RH-1-Induced Signals Leading the Wild-Type p53-Bearing Lung Cancer A549 Cells to Apoptosis.

Aziridinylquinone RH-1 (2,5-diaziridinyl-3-hydroxymethyl-6-methyl-cyclohexa-2,5-diene-1,4-dione) is a potential anticancer agent. RH-1 action is associated with NAD(P)H: quinone oxidoreductase (NQO1) which reduces this diaziridinylbenzoquinone into DNA-alkylating hydroquinone and is overexpressed in many tumors. Another suggested mechanism of RH-1 toxicity is the formation of reactive oxygen species (ROS) arising from its redox cycling. In order to improve anticancer action of this and similar antitumor quinones, we investigated the involvement of different signaling molecules in cytotoxicity induced by RH-1 by using wild-type tumor suppressor p53 bearing nonsmall cell lung carcinoma A549 cells as a model. Gradual and prolonged increase of mitogen-activated protein kinases (MAPK) ERK, P38, and JNK phosphorylation was observed during 24-h RH-1 treatment. In parallel, activation of DNA damage-sensing ATM kinase, upregulation, and phosphorylation of TP53 (human p53) took place. Inhibition studies revealed that RH-1-induced A549 apoptosis involved the NQO1-ATM-p53 signaling pathway and ROS generation. TP53 participated in ROS- and DNA damage-induced cell death differently. Moreover, MAP kinase JNK was another TP53 activator and death inducer in A549 cells. At the same time, rapid and prolonged activation of AKT kinase during RH-1 treatment was found, and it proved to be antiapoptotic kinase in our model system. Therefore, we identified that different and opposite cell death regulating signaling pathways, which may counteract one another, are induced in cancer cells during chemotherapeutic RH-1 treatment.

Identification of novel nitroreductases from Bacillus cereus and their interaction with the CB1954 prodrug.

Directed enzyme prodrug therapy is a form of cancer chemotherapy in which bacterial prodrug-activating enzymes, or their encoding genes, are directed to the tumour before administration of a prodrug. The prodrug can then be activated into a toxic drug at the tumour site, reducing off-target effects. The bacterial nitroreductases are a class of enzymes used in this therapeutic approach and although very promising, the low turnover rate of prodrug by the most studied nitroreductase enzyme, NfnB from Escherichia coli (NfnB_Ec), is a major limit to this technology. There is a continual search for enzymes with greater efficiency, and as part of the search for more efficient bacterial nitroreductase enzymes, two novel enzymes from Bacillus cereus (strain ATCC 14579) have been identified and shown to reduce the CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) prodrug to its respective 2'-and 4'-hydroxylamine products. Both enzymes shared features characteristic of the nitro-FMN-reductase superfamily including non-covalently associated FMN, requirement for the NAD(P)H cofactor, homodimeric, could be inhibited by Dicoumarol (3,3'-methylenebis(4-hydroxy-2H-chromen-2-one)), and displayed ping pong bi bi kinetics. Based on the biochemical characteristics and nucleotide alignment with other nitroreductase enzymes, one enzyme was named YdgI_Bc and the other YfkO_Bc. Both B. cereus enzymes had greater turnover for the CB1954 prodrug compared with NfnB_Ec, and in the presence of added NADPH cofactor, YfkO_Bc had superior cell killing ability, and produced mainly the 4'-hydroxylamine product at low prodrug concentration. The YfkO_Bc was identified as a promising candidate for future enzyme prodrug therapy.

Synthesis of 2-aryl-3-(2-cyanoethyl)aziridines and their chemical and enzymatic hydrolysis towards γ-lactams and γ-lactones.

Trans- and cis-2-aryl-3-(2-cyanoethyl)aziridines, prepared via alkylation of the corresponding 2-aryl-3-(tosyloxymethyl)aziridines with the sodium salt of trimethylsilylacetonitrile, were transformed into variable mixtures of 4-[aryl(alkylamino)methyl]butyrolactones and 5-[aryl(hydroxy)methyl]pyrrolidin-2-ones via KOH-mediated hydrolysis of the cyano group, followed by ring expansion. In addition, next to this chemical approach, enzymatic hydrolysis of the former aziridinyl nitriles by means of a nitrilase was performed as well, interestingly providing a selective route towards the above-mentioned functionalized γ-lactams.

A sensitive gel-based method combining distinct cyclophellitol-based probes for the identification of acid/base residues in human retaining ß-glucosidases.

Retaining ß-exoglucosidases operate by a mechanism in which the key amino acids driving the glycosidic bond hydrolysis act as catalytic acid/base and nucleophile. Recently we designed two distinct classes of fluorescent cyclophellitol-type activity-based probes (ABPs) that exploit this mechanism to covalently modify the nucleophile of retaining ß-glucosidases. Whereas ß-epoxide ABPs require a protonated acid/base for irreversible inhibition of retaining ß-glucosidases, ß-aziridine ABPs do not. Here we describe a novel sensitive method to identify both catalytic residues of retaining ß-glucosidases by the combined use of cyclophellitol ß-epoxide- and ß-aziridine ABPs. In this approach putative catalytic residues are first substituted to noncarboxylic amino acids such as glycine or glutamine through site-directed mutagenesis. Next, the acid/base and nucleophile can be identified via classical sodium azide-mediated rescue of mutants thereof. Selective labeling with fluorescent ß-aziridine but not ß-epoxide ABPs identifies the acid/base residue in mutagenized enzyme, as only the ß-aziridine ABP can bind in its absence. The Absence of the nucleophile abolishes any ABP labeling. We validated the method by using the retaining ß-glucosidase GBA (CAZy glycosylhydrolase family GH30) and then applied it to non-homologous (putative) retaining ß-glucosidases categorized in GH1 and GH116: GBA2, GBA3, and LPH. The described method is highly sensitive, requiring only femtomoles (nanograms) of ABP-labeled enzymes.

Broad-range glycosidase activity profiling.

Plants produce hundreds of glycosidases. Despite their importance in cell wall (re)modeling, protein and lipid modification, and metabolite conversion, very little is known of this large class of glycolytic enzymes, partly because of their post-translational regulation and their elusive substrates. Here, we applied activity-based glycosidase profiling using cell-permeable small molecular probes that react covalently with the active site nucleophile of retaining glycosidases in an activity-dependent manner. Using mass spectrometry we detected the active state of dozens of myrosinases, glucosidases, xylosidases, and galactosidases representing seven different retaining glycosidase families. The method is simple and applicable for different organs and different plant species, in living cells and in subproteomes. We display the active state of previously uncharacterized glycosidases, one of which was encoded by a previously declared pseudogene. Interestingly, glycosidase activity profiling also revealed the active state of a diverse range of putative xylosidases, galactosidases, glucanases, and heparanase in the cell wall of Nicotiana benthamiana. Our data illustrate that this powerful approach displays a new and important layer of functional proteomic information on the active state of glycosidases.

Spores of Clostridium engineered for clinical efficacy and safety cause regression and cure of tumors in vivo.

Spores of some species of the strictly anaerobic bacteria Clostridium naturally target and partially lyse the hypoxic cores of tumors, which tend to be refractory to conventional therapies. The anti-tumor effect can be augmented by engineering strains to convert a non-toxic prodrug into a cytotoxic drug specifically at the tumor site by expressing a prodrug-converting enzyme (PCE). Safe doses of the favored prodrug CB1954 lead to peak concentrations of 6.3 µM in patient sera, but at these concentration(s) known nitroreductase (NTR) PCEs for this prodrug show low activity. Furthermore, efficacious and safe Clostridium strains that stably express a PCE have not been reported. Here we identify a novel nitroreductase from Neisseria meningitidis, NmeNTR, which is able to activate CB1954 at clinically-achievable serum concentrations. An NmeNTR expression cassette, which does not contain an antibiotic resistance marker, was stably localized to the chromosome of Clostridium sporogenes using a new integration method, and the strain was disabled for safety and containment by making it a uracil auxotroph. The efficacy of Clostridium-Directed Enzyme Prodrug Therapy (CDEPT) using this system was demonstrated in a mouse xenograft model of human colon carcinoma. Substantial tumor suppression was achieved, and several animals were cured. These encouraging data suggest that the novel enzyme and strain engineering approach represent a promising platform for the clinical development of CDEPT.

Modulation of polyplex release from biodegradable microparticles through poly(ethylenimine) modification and varying loading concentration.

PURPOSE: This work investigates the effects of hyaluronic acid (HA) conjugated onto branched poly(ethylenimine) (bPEI) and varying loading concentrations of these polymers complexed with DNA on their release from poly(DL-lactic-co-glycolic acid) (PLGA) microparticles and the transfection of target cells. METHODS: To examine the effect of alteration of the gene delivery polymer on the system, we observed the morphology, size, loading efficiency, polymer and DNA release, and the transfection efficiency for the microparticles formed with three internal phase loading concentrations during microparticle formation. RESULTS: Addition of HA to this vector allowed for increased loading concentration within these systems and significantly altered release kinetics without changing the morphology of the particles. The incorporation of HA onto the bPEI backbone significantly increased the transfection efficiency of the complexes released from the corresponding microparticle formulation. CONCLUSIONS: The results show that the modification of bPEI with HA and the concentration of loaded polymer/DNA complexes can significantly alter the entrapment and release profiles from PLGA microparticles. This is significant in that it offers insight into the effects of modification of gene delivery vectors on a controlled release system designed to achieve a sustained therapeutic response.

Single-electron reduction of quinone and nitroaromatic xenobiotics by recombinant rat neuronal nitric oxide synthase.

We examined the kinetics of single-electron reduction of a large number of structurally diverse quinones and nitroaromatic compounds, including a number of antitumour and antiparasitic drugs, and nitroaromatic explosives by recombinant rat neuronal nitric oxide synthase (nNOS, EC 1.14.13.39), aiming to characterize the role of nNOS in the oxidative stress-type cytotoxicity of the above compounds. The steady-state second-order rate constants (kcat/Km) of reduction of the quinones and nitroaromatics varied from 10² M⁻¹s⁻¹ to 106 M⁻¹s⁻¹, and increased with an increase in their single-electron reduction potentials (E¹7). The presence of Ca²âº/calmodulin enhanced the reactivity of nNOS. These reactions were consistent with an 'outer sphere' electron-transfer mechanism, considering the FMNH∙/FMNH2 couple of nNOS as the most reactive reduced enzyme form. An analysis of the reactions of nNOS within the 'outer sphere' electron-transfer mechanism gave the approximate values of the distance of electron transfer, 0.39-0.47 nm, which are consistent with the crystal structure of the reductase domain of nNOS. On the other hand, at low oxygen concentrations ([O2] = 40-50 µM), nNOS performs a net two-electron reduction of quinones and nitroaromatics. This implies that NOS may in part be responsible for the bioreductive alkylation by two-electron reduced forms of antitumour aziridinyl-substituted quinones under a modest hypoxia.

Creation and screening of a multi-family bacterial oxidoreductase library to discover novel nitroreductases that efficiently activate the bioreductive prodrugs CB1954 and PR-104A.

Two potentially complementary approaches to improve the anti-cancer strategy gene-directed enzyme prodrug therapy (GDEPT) are discovery of more efficient prodrug-activating enzymes, and development of more effective prodrugs. Here we demonstrate the utility of a flexible screening system based on the Escherichia coli SOS response to evaluate novel nitroreductase enzymes and prodrugs in concert. To achieve this, a library of 47 candidate genes representing 11 different oxidoreductase families was created and screened to identify the most efficient activators of two different nitroaromatic prodrugs, CB1954 and PR-104A. The most catalytically efficient nitroreductases were found in the NfsA and NfsB enzyme families, with NfsA homologues generally more active than NfsB. Some members of the AzoR, NemA and MdaB families also exhibited low-level activity with one or both prodrugs. The results of SOS screening in our optimised E. coli reporter strain SOS-R2 were generally predictive of the ability of nitroreductase candidates to sensitise E. coli to CB1954, and of the kcat/Km for each prodrug substrate at a purified protein level. However, we also found that not all nitroreductases express stably in human (HCT-116 colon carcinoma) cells, and that activity at a purified protein level did not necessarily predict activity in stably transfected HCT-116. These results highlight a need for all enzyme-prodrug partners for GDEPT to be assessed in the specific context of the vector and cell line that they are intended to target. Nonetheless, our oxidoreductase library and optimised screens provide valuable tools to identify preferred nitroreductase-prodrug combinations to advance to preclinical evaluation.

Neurosteroid analog photolabeling of a site in the third transmembrane domain of the ß3 subunit of the GABA(A) receptor.

Accumulated evidence suggests that neurosteroids modulate GABA(A) receptors through binding interactions with transmembrane domains. To identify these neurosteroid binding sites directly, a neurosteroid-analog photolabeling reagent, (3α,5ß)-6-azi-pregnanolone (6-AziP), was used to photolabel membranes from Sf9 cells expressing high-density, recombinant, His(8)-ß3 homomeric GABA(A) receptors. 6-AziP inhibited (35)S-labeled t-butylbicyclophosphorothionate binding to the His(8)-ß3 homomeric GABA(A) receptors in a concentration-dependent manner (IC(50) = 9 ± 1 µM), with a pattern consistent with a single class of neurosteroid binding sites. [(3)H]6-AziP photolabeled proteins of 30, 55, 110, and 150 kDa, in a concentration-dependent manner. The 55-, 110-, and 150-kDa proteins were identified as His(8)-ß3 subunits through immunoblotting and through enrichment on a nickel affinity column. Photolabeling of the ß3 subunits was stereoselective, with [(3)H]6-AziP producing substantially greater labeling than an equal concentration of its diastereomer [(3)H](3ß,5ß)-6-AziP. High-resolution mass spectrometric analysis of affinity-purified, 6-AziP-labeled His(8)-ß3 subunits identified a single photolabeled peptide, ALLEYAF-6-AziP, in the third transmembrane domain. The identity of this peptide and the site of incorporation on Phe301 were confirmed through high-resolution tandem mass spectrometry. No other sites of photoincorporation were observed despite 90% sequence coverage of the whole ß3 subunit protein, including 84% of the transmembrane domains. This study identifies a novel neurosteroid binding site and demonstrates the feasibility of identifying neurosteroid photolabeling sites by using mass spectrometry.