The 1.5-? Structure of XplA-heme,an Unusual Cytochrome P450 Heme Domain That Catalyzes Reductive Biotransformation of Royal Demolition Explosive

XplA is a cytochrome P450 of unique structural organization, consisting of a heme- domain that is C-terminally fused to its native flavodoxin redox partner. XplA, along with flavodoxin reductase XplB, has been shown to catalyze the breakdown of the nitramine explosive and pollutant hexahydro-1,3,5-trinitro-1,3,5-triazine (royal demolition explosive) by reductive denitration. The structure of the heme domain of XplA (XplA-heme) has been solved in two crystal forms: as a dimer in space group P2₁ to a resolution of 1.9 Å and as a monomer in space group P2₁2₁2 to a resolution of 1.5 Å, with the ligand imidazole bound at the heme iron. Although it shares the overall fold of cytochromes P450 of known structure, XplA-heme is unusual in that the kinked I-helix that traverses the distal face of the heme is broken by Met-394 and Ala-395 in place of the well conserved Asp/Glu plus Thr/Ser, important in oxidative P450s for the scission of the dioxygen bond prior to substrate oxygenation. The heme environment of XplA-heme is hydrophobic, featuring a cluster of three methionines above the heme, including Met-394. Imidazole was observed bound to the heme iron and is in close proximity to the side chain of Gln-438, which is situated over the distal face of the heme. Imidazole is also hydrogen-bonded to a water molecule that sits in place of the threonine side-chain hydroxyl exemplified by Thr-252 in Cyt-P450cam. Both Gln-438 → Ala and Ala-395 → Thr mutants of XplA-heme displayed markedly reduced activity compared with the wild type for royal demolition explosive degradation when combined with surrogate electron donors.Royal demolition explosive (RDX)² or cyclotrimethylenetrinitramine 1 (see Fig. 1) is a widely used explosive compound with both military and civil applications. The extensive global usage of RDX has resulted in concerns over environmental contamination, because it is both recalcitrant to degradation, leading to contamination in soil and ground water, and a potent convulsant and possible carcinogen. The bioremediation of RDX has thus been the focus of increasing research in recent years, with a number of bacterial strains reported to catalyze its degradation (1–3). Among these, Bruce and co-workers, using a selective enrichment technique, isolated Rhodococcus rhodochrous strain 11Y from an RDX-contaminated site, which was able to grow on RDX as the sole nitrogen source (4). The products of biotransformation of RDX by this bacterium were shown to be nitrite and formaldehyde. A gene cluster in strain 11Y essential for RDX degradation was identified and shown to contain a novel cytochrome P450, termed XplA, and a redox partner, XplB, which were shown together to be capable of catalyzing the biotransformation of RDX in vitro (5). Near identical xplA and xplB genes have now been identified in strains within the Actinomycetales isolated from geographically distinct sites (6). Interestingly, these genes have been found to be plasmid encoded providing compelling evidence for recent lateral gene transfer. In the interests of developing an efficient technology for the targeted phytoremediation of RDX, the XplA-XplB system was expressed in transgenic strains of Arabidopsis thaliana and shown to successfully remediate RDX from contaminated soil (5, 7).Open in a separate windowFIGURE 1.Biocatalyzed routes proposed for the degradation of the nitramine exposive RDX 1 by cytochrome P450 XplA from Rhodococcus rhodochrous 11Y. Pathway A corresponds to the route proposed under anaerobic conditions to give the cleavage product metabolite methylene dinitramine 6; pathway B is that postulated to occur under aerobic conditions to give cleavage product 4-nitro-2,4-diazabutanal 7.RDX has an unusual chemical structure, featuring three nitramine (N-NO₂) bonds on a saturated six-membered ring, a functionality that has few precedents among natural products (8). The products isolated from XplA-XplB-catalyzed degradation of RDX in vitro were shown to be different under aerobic and anaerobic conditions after initial denitration to imine intermediate 2 (Fig. 1) (5). Under anaerobic conditions A, 2 would undergo hydration to give intermediate 4. Ring cleavage would yield the isolable metabolite methylene dinitramine 6. The aerobic process B was thought to proceed via successive denitration of two nitramine groups, to give the di-imine 3, which would then be twice hydrated to give the unstable intermediate 5, which would undergo spontaneous ring cleavage to give the isolable product 4-nitro-2,4-diazabutanal 7, formaldehyde, and two equivalents of nitrite.Although the reductive denitration of, for example, glycerol trinitrate (9) and other nitroheterocylic drugs (10), has previously been described for mammalian cytochromes P450, the best known reactions catalyzed by this family of enzymes include a wide range of oxidative chemical reactions, including hydroxylation, heteroatom dealkylation, and C–C bond cleavage reactions most commonly using molecular oxygen and involving cleavage of the O–O bond after dioxygen is bound by the heme-iron (11). A sequence alignment of some cyt-P450 for which the structures have been determined (Fig. 2) shows that the well conserved residues Glu/Asp-Thr/Ser that are thought to be a requirement for oxidative chemistry (12, 13) and to aid cleavage of the scissile O–O bond are not conserved in XplA, being replaced by methionine-394 and alanine-395. In the interests of illuminating the molecular mechanism and substrate specificity in XplA, we were thus interested in obtaining a structure of the heme domain of the enzyme. In this report, the structure of XplA-heme domain in two crystal forms is presented: the first with two molecules in the asymmetric unit, derived from the attempted crystallization of full-length XplA and the other derived from the subcloned, isolated XplA-heme domain, with one molecule in the asymmetric unit and the heme iron ligated to the substrate analogue imidazole. The results reveal a possible access channel for ligand transport and, in combination with mutational studies, demonstrate a highly unusual active site environment for substrate binding, suggesting substrate binding or catalytic roles for Gln-438 and Ala-395.Open in a separate windowFIGURE 2.Amino acid sequence alignment for portions of selected CYP heme domains for which the structures have been solved. P450_pikC (from Streptomyces venezuelae; Uniprot accession no. {"type":"entrez-protein","attrs":{"text":"O87605","term_id":"75539043","term_text":"O87605"}}O87605); P450_cin (from Citrobacter braakii; {"type":"entrez-protein","attrs":{"text":"Q8VQ86","term_id":"75529099","term_text":"Q8VQ86"}}Q8VQ86); P450_eryF (Saccharopolyspora erythrea; {"type":"entrez-protein","attrs":{"text":"Q00441","term_id":"729202","term_text":"Q00441"}}Q00441); P450_moxa (Nonomuraea recticatena; {"type":"entrez-protein","attrs":{"text":"Q2L6S8","term_id":"123293472","term_text":"Q2L6S8"}}Q2L6S8); P450_staP (Streptomyces sp. TP-A0274; {"type":"entrez-protein","attrs":{"text":"Q83WG3","term_id":"81321418","term_text":"Q83WG3"}}Q83WG3); P450_BioI (B. subtilis; {"type":"entrez-protein","attrs":{"text":"P53554","term_id":"1705469","term_text":"P53554"}}P53554); P450_epok (Sorangium cellulosum; {"type":"entrez-protein","attrs":{"text":"Q9KIZ4","term_id":"41688519","term_text":"Q9KIZ4"}}Q9KIZ4); P450_terp (Pseudomonas sp.; {"type":"entrez-protein","attrs":{"text":"P33006","term_id":"416837","term_text":"P33006"}}P33006); P450_xpla (Rhodococcus sp. 11Y; {"type":"entrez-protein","attrs":{"text":"Q8GPH7","term_id":"81322300","term_text":"Q8GPH7"}}Q8GPH7); P450_cam (Pseudomonas putida; {"type":"entrez-protein","attrs":{"text":"P00183","term_id":"117297","term_text":"P00183"}}P00183); and P450_BM3 (Bacillus megaterium; {"type":"entrez-protein","attrs":{"text":"P14779","term_id":"117298","term_text":"P14779"}}P14779). Each catalyzes oxidative chemistry and most apart from P450_cin contain the (E/D)(S/T) dyad (shown in red) demonstrated to be important in the scission of the dioxygen bond as a prerequisite of substrate mono-oxygenation. XplA possesses Met-394 and Ala-395 (shown in green).     

Lack of effect of methylene blue in the SOD1 G93A mouse model of amyotrophic lateral sclerosis

Background

Methylene blue (MB) is a drug with a long history and good safety profile, and with recently-described features desirable in a treatment for ALS.

Methodology/Principal Findings

We tested oral MB in inbred high-copy number SOD1 G93A mice, at 25 mg/kg/day beginning at 45 days of age. We measured disease onset, progression, and survival. There was no difference in disease onset between MB-treated mice and controls, although subgroup analysis showed a modest but statistically significant delay in disease onset in MB-treated female mice only (control 122±10.2 versus MB 129±10.0 days). MB-treated mice of both sexes spent more time in less severe stages of disease, and less time in later, more severe stages of disease. There was a non-significant trend to longer survival in MB-treated animals (control males reached endpoint at 161±14.1 days, versus 166±10.0 days for MB-treated animals, and control females reached endpoint at 171±6.2 days versus 173±13.4 days for MB-treated animals).

Conclusions/Significance

In spite of a strong theoretical rationale, MB had no significant effects on onset or survival in the inbred SOD1 G93A mouse model of ALS.     

Tropical rain forest conversion and perspectives in the conservation of wild primates (Alouatta and Ateles) in Mexico

The original distribution of the tropical rain forest and of the populations of Alouatta palliata, Al. pigra, and the two subspecies of Ateles geoffroyi in southern Mexico have been reduced by at least 90% in the last 40 years as a result of conversion of natural habitat to pasture and agricultural fields. This dramatic conversion has been caused mainly by the rapid growth of the human population in the southern states of the country. In the region of Los Tuxtlas in southern Veracruz, where the only longitudinal popululational and ecological studies of A. palliata and At. g. vellerosus have taken place, only 15% of the original extension of the tropical rain forest remains today. The intensive destruction of suitable primate habitat in this region has resulted in an accelerated process of extinction of the primate species. It is estimated that only about 200 At. g. vellerosus and about 1200 Al. palliata exist in the remaining small portion of their original habitat. Today, the distribution of the three primate species in Mexico is intensively and extensively fragmented, and only five potential foci for conservation exist in the country. Urgent action is required to protect the primate populations in the region of Los Tuxtlas and at the other four foci, as some of these populations may disappear by 1995.     

A preliminary study of resource overlap between howling monkeys (Alouatta palliata) and other arboreal mammals in the tropical rain forest of Los Tuxtlas,Mexico

Potential resource overlap between howling monkeys and other arboreal mammals was studied in the rain forest of Los Tuxtlas, Veracruz, Mexico. Eight species of mammals belonging to the orders Primates, Carnivora, Rodentia, and Marsupialia were found to share the canopy and to overlap trophically with howling monkeys. These mammals made up 77% and Alouatta 23% of the arboreal mammalian biomass under consideration. The arboreal porcupine and spider monkey were the only mammals that also fed on leaves. However, in this feeding niche, Alouatta is the only important mammalian folivore in Los Tuxtlas, and resource depression derived from leaf-eating insects is more important. The eight arboreal mammals may exert more pressure upon fruit resources, for they consumed 75% of the estimated total dry weight of fruit/ha/yr consumed by arboreal mammals.     

Bird species richness in vegetation fences and in strips of residual rain forest vegetation at Los Tuxtlas, Mexico

Fragmentation of the lowland tropical rain forest has resulted in loss of animal and plant species and isolation of remaining populations that puts them at risk. At Los Tuxtlas, Mexico, lowland rain forests are particularly diverse in the avian fauna they contain and while most of the forests have been fragmented by human activity, many of the fragments still harbor diverse assemblages of bird species. In these landscapes, linear strips of residual rain forest vegetation along streams as well as linear strips of vegetation fences (live fences) crossing the pastures might provide some connectivity to bird populations existed in forest fragments. We investigated bird species richness and relative abundance in one 6-km long section of live fences (LF) bordering a dirt road and in two 6-km long sections of residual forest vegetation along a river (MR) and one permanent stream (BS). We used point count procedures which resulted in the count of 2984 birds representing 133 species. At the LF site we detected 74% of the species, 72% at the BS site and 57% at the MR site. Only 38% of the species were common among sites. Neotropical migratory birds accounted for 34–41% of the species counted at all sites. While edge and open habitat birds accounted for 6–10% of the species and for 50% of the records at the three vegetation strips, about 90% of the species were forest birds. Distance to forest fragments and degree of disturbance of the vegetation seemed to negatively influence bird species presence at the BS and MR strips. Rarefaction analysis indicated that the LF strip was richer in species than the other two sites, but the occurrence of the three vegetation strips in the landscape seem to favor the presence of many more species. We discuss the value of these vegetation strips to birds as stepping stones in the fragmented landscape.     

Tropical rain forest fragmentation and wild populations of primates at Los Tuxtlas,Mexico

In view of the extensive destruction, fragmentation, and conversion of primate habitats to anthropogenic vegetation, information on Neotropical primate ability to use a landscape consisting of forest fragments and agricultural habitats is necessary to understand the ecological flexibility of the species involved and it is of relevance to the design of conservation scenarios at the landscape level. We censused howlers and spider monkeys in 126 forest fragments and in 44 agricultural sites at Los Tuxtlas, southern Veracruz, Mexico, and used the IDRISI Geographic Information System to assess the extent of primate habitat remaining. We conducted economic surveys to assess the productivity of several systems of land management, including cattle ranching. Seventy- five percent of native habitat has been lost at Los Tuxtlas, 20% remains in the form of isolated fragments of vegetation, and only 5% consists of contiguous rain forest at high elevations (> 800 m). Howlers and spider monkeys were present in only 60 and 8% of the forest sites investigated, respectively, attesting to extensive local extinction. Populations of both species are small and found in a fragmented and isolated condition throughout the landscape. A large number of howlers were detected in artifactual habitats such as cacao, coffee and mixed (cacao and coffee) plantations shaded by rain forest trees. Residual rain forest vegetation along streams, rivers, and lakes facilitated the interfragment and interhabitat movement of howlers. Economic surveys showed that some of the agricultural habitats were more productive than cattle ranching. Conservation of spider monkeys requires large areas of contiguous forest, but only small areas are present at high elevations. Howlers are restricted to elevations < 800 m, where most of the forest has disappeared, but can apparently exist in a matrix of forest fragments, arboreal agricultural habitats, and pasture lands. Structural aspects of the vegetation and connectivity among habitat islands may be indispensable components of both landscape scenarios.     

Salicylate nephropathy in the Gunn rat: Potential role of prostaglandins

We examined the potential role of prostaglandins in the development of analgesic nephropathy in the Gunn strain of rat. The homozygous Gunn rats have unconjugated hyperbilirubinemia due to the abscence of glucuronyl transferase, leading to marked bilirubin deposition in renal medulla and papilla. These rats are also highly susceptible to develop papillary necrosis with analgesic administration.We used homozygous (jj) and phenotypically normal heterozygous )jJ) animals. Four groups of rats (n = 7) were studied: jj and jJ rats treated either with aspirin 300 mg/kg every other day or sham-treated. After one week, slices of cortex, outer and inner medulla from one kidney wre incubated in buffer and prostaglandin synthesis was determined by radioimmunoassay. The other kidney was examined histologically.A marked corticomedullary gradient of prostaglandin synthesis was observed in all groups, PGE2 synthesis was significantly higher in outer medulla, but not cortex or inner medulla, of jj (38 ± 6 mg/mg prot) than jJ rats (15 ± 3) (p<0.01). Aspirin treatment reduced PGE2 synthesis in all regions, but outer medullary PGE2 remained higher in jj (18 ± 3) than jJ rats (9 ± 2) (p<0.05). PGE2α was also significantly higher in the outer medulla of jj rats with and without aspirin administration (p<0.05). The changes in renal prostaglandin synthesis were accompanied by evidence of renal damage in aspirin-treated jj but not jJ rats as evidenced by: increased incidence and severity of hematuria (p<0.01); increased serum creatinine (p<0.05); and increase in outer medullary histopathologic lesions (p<0.005 compared to either sham-treated jj or aspirin-treated jJ).These results suggest that enhanced protaglandin synthesis contributes to maintenance of renal function and morphological integrity, and that inhibition of protaglandin synthesis may lead to pathological renal medullary lesions and deterioration of renal function.