Brevibacillus laterosporus strain BPM3, a potential biocontrol agent isolated from a natural hot water spring of Assam, India

A bacterial strain designated as BPM3 isolated from mud of a natural hot water spring of Nambar Wild Life Sanctuary, Assam, India, strongly inhibited growth of phytopathogenic fungi (Fusarium oxysporum f. sp. ciceri, F. semitectum, Magnaporthe grisea and Rhizoctonia oryzae) and gram-positive bacterium (Staphylococcus aureus). The maximum growth and antagonistic activity was recorded at 30 °C, pH 8.5 when starch and peptone were amended as carbon and nitrogen sources, respectively. In greenhouse experiment, this bacterium (BPM3) suppressed blast disease of rice by 30-67% and protected the weight loss by 35-56.5%. The maximum disease protection (67%) and weight loss protection (56.5%) were recorded when the bacterium was applied before 2 days of the pathogen inoculation. Antifungal and antibacterial compounds were isolated from the bacterium which also inhibited the growth of these targeted pathogens. The compounds were purified and on spectroscopic analysis of a purified fraction having R_f 0.22 which showed strong antifungal and antibacterial activity indicated the presence of C-H, carbonyl group, dimethyl group, -CH₂ and methyl group. The bacterium was characterized by morphological, biochemical and molecular approaches and confirmed that the strain BPM3 is Brevibacillus laterosporus.     

Study on the biochemical basis of mefloquine resistant Plasmodium falciparum

Increase in drug detoxification and alteration of drug uptake and efflux of Plasmodium falciparum were investigated for their possible association with mefloquine (MQ) resistance in five different clones of P. falciparum from Thailand (T994b₃, K1CB₂, PR70CB₁, PR71CB₂ and TM₄CB8-2.2.3). Fifty percent inhibitory concentration (IC₅₀) values from these five clones varied between 30- and 50-fold. Regarding the detoxification mechanism, the ability of P. falciparum clones to biotransform MQ was shown in vitro by parasite microsomal protein prepared from parasite infected red blood cells protein (30 μg), NADPH (1 nM) and phosphate buffer pH 7.4, carried out at 37 °C with agitation. Radiolabelled unmetabolized MQ and possible metabolite(s) generated from the reaction was extracted into ethylacetate and separated by radiometric-HPLC after 1 h. All clones were capable of converting MQ into carboxymefloquine (CMQ), which is the main metabolite in human plasma. In addition, another unidentified metabolite eluted at 4.2 min on the chromatograph could be detected from the incubation reaction. This metabolite has never been detected in human liver microsomes before. There was no significant difference in the percentages of CMQ formed in the resistant (T994_b3, PR₇₀CB₁, PR₇₁CB₂) and sensitive (TM₄CB8-2.2.3, K1CB₂) clones. Another possible mechanism, i.e., alteration in the accumulation of MQ in the parasites was investigated in vitro using [¹⁴C]MQ as a tracer. The time courses of [¹⁴C]MQ uptake and efflux were generally characterized by two phases. A trend of increased efflux of [¹⁴C]MQ was observed in the resistant compared with sensitive clones.     

Acrosome reaction is subfamily specific in sea star fertilization

In the fertilization process of sea stars, sperm is activated to go through the acrosome reaction before cell fusion. We focused on induction of the acrosome reaction as a key process in fertilization. Six species of sea stars were used in this study: Asterias amurensis, Asterias rubens, Asterias forbesi, Aphelasterias japonica, Distolasterias nipon, and Asterina pectinifera. Acrosome reaction assays indicate that the acrosome reaction can be induced across species within Asteriinae subfamily. However, cross-fertilization assays indicate that sea stars have species specificity in fertilization. Therefore, steps after the acrosome reaction are responsible for the species specificity. To explain acrosome reaction subfamily specificity at the molecular level, the sugar components of egg jelly were examined and analyzed by principal component analysis. A. amurensis and A. forbesi belong to the same induction group of the acrosome reaction. D. nipon and An. pectinifera are in a unique group. Enzyme-linked immunosorbent assays indicate that Asteriinae subfamily share a common glycan structure, the Fragment 1 of Acrosome Reaction-Inducing Substance from A. amurensis. Fragment 1 plays an important role in the subfamily specificity of acrosome reaction induction. In addition, A. amurensis sperm activating peptide was recognized by sperm from the same superorder. These results demonstrate that the specificity of acrosome reaction induction is present at the subfamily level in sea stars.     

Synthesis and anti-tumor activity of novel ethyl 3-aryl-4-oxo-3,3a,4,6-tetrahydro-1H-furo[3,4-c]pyran-3a-carboxylates

A series of ethyl 3-aryl-4-oxo-3,3a,4,6-tetrahydro-1H-furo[3,4-c]pyran-3a-carboxylates were prepared through the metal-catalyzed domino reaction of alkylidene malonates and 1,4-butynediol under a one-pot reaction condition at room temperature. Their in vitro anti-proliferative activities were subsequently evaluated in A549, QGY and HeLa cells. The majority of the compounds showed potent anti-tumor activity against HeLa cells. In particular, compound 3l was the most potent compound with IC₅₀ value of 5.4 μM. For the first time, the X-ray structure of the anti-tumor ethyl 3-aryl-4-oxo-3,3a,4,6-tetrahydro-1H-furo[3,4-c]pyran-3a-carboxylates is determined.     

Vibrios dominate as culturable nitrogen-fixing bacteria of the Brazilian coral Mussismilia hispida

Taxonomic characterization was performed on the putative N₂-fixing microbiota associated with the coral species Mussismilia hispida, and with its sympatric species Palythoa caribaeorum, P. variabilis, and Zoanthus solanderi, off the coast of São Sebastião (São Paulo State, Brazil). The 95 isolates belonged to the Gammaproteobacteria according to the 16S rDNA gene sequences. In order to identify the isolates unambiguously, pyrH gene sequencing was carried out. The majority of the isolates (n=76) fell within the Vibrio core group, with the highest gene sequence similarity being towards Vibrio harveyi and Vibrio alginolyticus. Nineteen representative isolates belonging to V. harveyi (n=7), V. alginolyticus (n=8), V. campbellii (n=3), and V. parahaemolyticus (n=1) were capable of growing six successive times in nitrogen-free medium and some of them showed strong nitrogenase activity by means of the acetylene reduction assay (ARA). It was concluded that nitrogen fixation is a common phenotypic trait among Vibrio species of the core group. The fact that different Vibrio species can fix N₂ might explain why they are so abundant in the mucus of different coral species.     

Experimental concurrent infection of Afar breed goats with Oestrus ovis (L1) and Haemonchus contortus (L3): interaction between parasite populations, changes in parasitological and basic haematological parameters

The study was conducted to examine the occurrence and interaction between Oestrus ovis and Haemonchus contortus in experimentally infected Ethiopian Afar breed of goats. Twenty goats were divided into four groups (O, OH, H, and C) of five animals each. Each animal of groups O and OH received weekly infections for 5 weeks with 66 first instar larvae (L₁) of O. ovis. Then animals of groups OH and H were infected with a single dose of 5000 third stage larvae (L₃) of H. contortus. Goats of group C were kept free of any infection as non-infected control. Faecal egg count (FEC), blood cell count, total serum protein level and body weight were recorded weekly throughout the study period. At necropsy worm burden, female worm length, fecundity and larval burden of O. ovis in the nasal-sinus cavities of infected animals were assessed. The results showed that the presence of H. contortus in the abomasum of goats of group OH had no influence on the development of O. ovis. On the contrary, a significant reduction (P < 0.05) in FEC, worm burden, fecundity and female worm length was revealed in group OH animals compared to the mono-infected animals (group H). This was associated with eosinophilia and reduced packed cell volume.     

Generation of primary amide glucosides from cyanogenic glucosides

The cyanogenic glucoside-related compound prunasinamide, (2R)-β-d-glucopyranosyloxyacetamide, has been detected in dried, but not in fresh leaves of the prunasin-containing species Olinia ventosa, Prunus laurocerasus, Pteridium aquilinium and Holocalyx balansae. Experiments with leaves of O. ventosa indicated a connection between amide generation and an excessive production of reactive oxygen species. In vitro, the Radziszewski reaction with H₂O₂ has been performed to yield high amounts of prunasinamide from prunasin. This reaction is suggested to produce primary amides from cyanogenic glycosides in drying and decaying leaves. Two different benzoic acid esters which may be connected to prunasin metabolism were isolated and identified as the main constituents of chlorotic leaves from O. ventosa and P. laurocerasus.     

Reconstitution in vitro of the catalytic portion (NtpA3-B3-D-G complex) of Enterococcus hirae V-type Na-ATPase

Enterococcus hirae vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain (V₁; NtpA₃-B₃-D-G) and an integral membrane domain (V₀; NtpI-K₁₀) connected by a central and peripheral stalk(s) (NtpC and NtpE-F). Here we examined the nucleotide binding of NtpA monomer, NtpB monomer or NtpD-G heterodimer purified by using Escherichia coli expression system in vivo or in vitro, and the reconstitution of the V₁ portion with these polypeptides. The affinity of nucleotide binding to NtpA was 6.6 μM for ADP or 3.1 μM for ATP, while NtpB or NtpD-G did not show any binding. The NtpA and NtpB monomers assembled into NtpA₃-B₃ heterohexamer in nucleotide binding-dependent manner. NtpD-G bound NtpA₃-B₃ forming V₁ (NtpA₃-B₃-D-G) complex independent of nucleotides. The V₁ formation from individual NtpA and NtpB monomers with NtpD-G heterodimer was absolutely dependent on nucleotides. The ATPase activity of reconstituted V₁ complex was as high as that of native V₁-ATPase purified from the V₀V₁ complex by EDTA treatment of cell membrane. This in vitro reconstitution system of E. hirae V₁ complex will be valuable for characterizing the subunit-subunit interactions and assembly mechanism of the V₁-ATPase complex.     

Development of multiplex PCR assay for rapid detection of Riemerella anatipestifer, Escherichia coli, and Salmonella enterica simultaneously from ducks

Three pathogens, Riemerella anatipestifer, Escherichia coli, and Salmonella enterica, are leading causes of bacterial fibrinous pericarditis and perihepatitis in ducks in China and worldwide. It is difficult to differentiate these pathogens when obtaining a diagnosis on clinical signs and pathological changes. The aim of this research was to develop a multiplex polymerase chain reaction (m-PCR) that could discriminate R. anatipestifer, E. coli, and S. enterica rapidly in field isolates, or detect the three bacteria in clinical samples from diseased ducks. We selected the DnaB helicase (dnaB) gene of R. anatipestifer, alkaline phosphatase (phoA) gene of E. coli and invasion protein (invA) gene of S. enterica as target genes. In optimized conditions, the limitation of detection was approximately 10³ colony forming units (CFU) of each of these three bacterial pathogens per PCR reaction tube. The m-PCR method showed specific amplification of respective genes from R. anatipestifer, E. coli, and S. enterica. Using the m-PCR system, bacterial strains isolated from diseased ducks in our laboratory were categorized successfully, and the pathogens could also be detected in clinical samples from diseased ducks. Therefore, the m-PCR system could distinguish the three pathogens simultaneously, for identification, routine molecular diagnosis and epidemiology, in a single reaction.     

Cyclical expression of the Notch/Wnt regulator Nrarp requires modulation by Dll3 in somitogenesis

Delta-like 3 (Dll3) is a divergent ligand and modulator of the Notch signaling pathway only identified so far in mammals. Null mutations of Dll3 disrupt cycling expression of Notch targets Hes1, Hes5, and Lfng, but not of Hes7. Compared with Dll1 or Notch1, the effects of Dll3 mutations are less severe for gene expression in the presomitic mesoderm, yet severe segmentation phenotypes and vertebral defects result in both human and mouse. Reasoning that Dll3 specifically disrupts key regulators of somite cycling, we carried out functional analysis to identify targets accounting for the segmental phenotype. Using microdissected embryonic tissue from somitic and presomitic mesodermal tissue, we identified new genes enriched in these tissues, including Limch1, Rhpn2, and A130022J15Rik. Surprisingly, we only identified a small number of genes disrupted by the Dll3 mutation. These include Uncx, a somite gene required for rib and vertebral patterning, and Nrarp, a regulator of Notch/Wnt signaling in zebrafish and a cycling gene in mouse. To determine the effects of Dll3 mutation on Nrarp, we characterized the cycling expression of this gene from early (8.5 dpc) to late (10.5 dpc) somitogenesis. Nrarp displays a distinct pattern of cycling phases when compared to Lfng and Axin2 (a Wnt pathway gene) at 9.5 dpc but appears to be in phase with Lfng by 10.5 dpc. Nrarp cycling appears to require Dll3 but not Lfng modulation. In Dll3 null embryos, Nrarp displayed static patterns. However, in Lfng null embryos, Nrarp appeared static at 8.5 dpc but resumed cycling expression by 9.5 and dynamic expression at 10.5 dpc stages. By contrast, in Wnt3a null embryos, Nrarp expression was completely absent in the presomitic mesoderm. Towards identifying the role of Dll3 in regulating somitogenesis, Nrarp emerges as a potentially important regulator that requires Dll3 but not Lfng for normal function.     

Fabacyl acetate, a germination stimulant for root parasitic plants from Pisum sativum

A germination stimulant, fabacyl acetate, was purified from root exudates of pea (Pisum sativum L.) and its structure was determined as ent-2′-epi-4a,8a-epoxyorobanchyl acetate [(3aR,4R,4aR,8bS,E)-4a,8a-epoxy-8,8-dimethyl-3-(((R)-4-methyl-5-oxo-2,5-dihydrofuran-2-yloxy)methylene)-2-oxo-3,3a,4,5,6,7,8,8b-decahydro-2H-indeno[1,2-b]furan-4-yl acetate], by 1D and 2D NMR spectroscopic, ESI- and EI-MS spectrometric, X-ray crystallographic analyses, and by comparing the ¹H NMR spectroscopic data and relative retention times (RR_t) in LC-MS and GC-MS with those of synthetic standards prepared from (+)-orobanchol and (+)-2′-epiorobanchol. The ¹H NMR spectroscopic data and RR_t of fabacyl acetate were identical with those of an isomer prepared from (+)-2′-epiorobanchol except for the opposite sign in CD spectra. This is the first natural ent-strigolactone containing an epoxide group. Fabacyl acetate was previously detected in root exudates of other Fabaceae plants including faba bean (Vicia faba L.) and alfalfa (Medicago sativa L.).     

Treatment with NaHSO3 greatly enhances photobiological H2 production in the green alga Chlamydomonas reinhardtii

Treatment with NaHSO₃ induces a 10-fold increase in H₂ photoproduction in the filamentous N₂-fixing cyanobacterium Anabaena sp. strain PCC 7120. However, it is unclear whether this treatment also increases H₂ photoproduction in green alga. In this study, treatment with 13 mM NaHSO₃ resulted in about a 200-fold increase in H₂ production in Chlamydomonas reinhardtii, and this increase was most probably the result of reduced O₂ content and enhanced hydrogenase activity. Compared to the conventional strategy of sulfur deprivation, NaHSO₃ treatment results in a higher maximum rate of H₂ photoproduction, greater efficiency of conversion of light energy into H₂, shorter half-time to produce the maximum accumulated H₂ levels, and reduced costs because no centrifugation is involved. We therefore conclude that NaHSO₃ treatment is an efficient, rapid, and economic strategy for improving photobiological H₂ production in the green alga C. reinhardtii.     

Effects of insect cuticular fatty acids on in vitro growth and pathogenicity of the entomopathogenic fungus Conidiobolus coronatus

Eighteen fatty acids identified in the cuticle of three insect species representing differing susceptibilities to C. coronatus infection, were tested for effects on the in vitro growth and pathogenicity of the parasitic fungus. At all applied concentrations (0.1-0.0001% w/v) growth was inhibited by C_16:0, C_16:1, C_18:0, C_18:1, C_18:2, C_18:3, C_20:0 and C_20:1. At high concentrations spore germination was inhibited by C_7:0, C_8:0, C_9:0, C_10:0, C_12:0, C_18:2 and C_18:3 and hyphal growth was merely retarded by C_5:0, C_6:0, C_6:2, C_14:0, C_16:0, C_16:1, C_18:0, C_18:1, C_20:0 and C_20:1. The presence of C_15:0 at the 0.1% concentration stimulated growth of C. coronatus. Sporulation was inhibited by all concentrations of C_16:0 and C_18-20 fatty acids. Low concentrations of C_5:0, C_6:0, C_6:2 and C_7:0 enhanced sporulation. Fatty acids C_5-12 as well as C_18:3, C_20:0 and C_20:1 decreased the ability of fungal colonies to infect G. mellonella while C_16:1 elevated it thus suggesting that C_16:1 may stimulate production of enzymes involved in the host invasion. Toxicity of metabolites released into incubation medium decreased with varying degrees in the presence of C_6:0, C_6:2, C_7:0, C_9:0, C_12:0, C_16:1, C_18:2, C_18:3, C_20:0 and C_20:1; other fatty acids had no effect. Further work is needed to analyse the effects of exogenous fatty acids on the C. coronatus enzymes implicated in fungal pathogenicity as well as on the production of insecticidal metabolites.     

Interactions between TGF-β1 and TGF-β3 and their role in medial edge epithelium cell death and palatal fusion in vitro

In recent decades, studies have shown that both TGF-β₁ and TGF-β₃ play an important role in the induction of medial edge epithelium (MEE) cell death and palatal fusion. Many of these experiments involved the addition or blockage of one of these growth factors in wild-type (WT) mouse palate cultures, where both TGF-β₁ and TGF-β₃ are present. Few studies have addressed the existence of interactions between TGF-β₁ and TGF-β₃, which could modify their individual roles in MEE cell death during palatal fusion. We carried out several experiments to test this possibility, and to investigate how this could influence TGF-β₁ and TGF-β₃ actions on MEE cell death and palatal shelf fusion. We double-immunolabelled developing mouse palates with anti-TGF-β₁ or anti-TGF-β₃ antibodies and TUNEL, added rhTGF-β₁ or rhTGF-β₃ or blocked the TGF-β₁ and TGF-β₃ action at different concentrations to WT or Tgf-β₃ null mutant palate cultures, performed in situ hybridizations with Tgf-β₁ or Tgf-β₃ riboprobes, and measured the presence of TUNEL-positive midline epithelial seam (MES) cells and MES disappearance (palatal shelf fusion) in the different in vitro conditions. By combining all these experiments, we demonstrate great interaction between TGF-β₁ and TGF-β₃ in the developing palate and confirm that TGF-β₃ has a more active role in MES cell death than TGF-β₁, although both are major inductors of MES disappearance. Finally, the co-localization of TGF-β₁, but not TGF-β₃, with TUNEL in the MES allows us to suggest a possible role for TGF-β₁ in MES apoptotic clearance.     

Rubrimonas shengliensis sp. nov. and Polymorphum gilvum gen. nov., sp. nov., novel members of Alphaproteobacteria from crude oil contaminated saline soil

Four bacterial strains were isolated from a crude oil contaminated saline soil in Shengli Oilfield, China. Strains SL014B-28A2^T and SL014B-80A1 were most closely related to Rubrimonas cliftonensis OCh 317^T, while strains SL003B-26A1^T and SL003B-26A2 were most closely related to but readily different from the species in the Pannonibacter-Labrenzia-Roseibium-Stappia cluster. The major fatty acids were C_18:1ω7c, C_16:0, C_18:0 and 11-Methyl C_18:1ω7c, and C_18:1ω7c, 11-Methyl C_18:1ω7c and C_18:0, respectively, for these two groups of isolates. Q-10 was the predominant ubiquinone. The G + C contents of genomic DNA of the four isolates were 67.9, 69.7, 65.6 and 65.6 mol%. Based on the polyphasic taxonomic characteristics, strains SL014B-28A2^T and SL014B-80A1 represented a novel species of the genus Rubrimonas, for which the name Rubrimonas shengliensis sp. nov. is proposed, with strain SL014B-28A2^T (=LMG 26072^T = CGMCC 1.9170^T) as the type strain. Isolates SL003B-26A1^T and SL003B-26A2 represented a novel genus and species of the family Rhodobacteraceae, for which the name Polymorphum gilvum gen. nov., sp. nov. is proposed, with strain SL003B-26A1^T (=LMG 25793^T = CGMCC 1.9160^T) as the type strain.     

Crystal structure of the Clostridium limosum C3 exoenzyme

C3-like toxins ADP-ribosylate and inactivate Rho GTPases. Seven C3-like ADP-ribosyltransferases produced by Clostridium botulinum, Clostridium limosum, Bacillus cereus and Staphylococcus aureus were identified and two representatives - C3bot from C. botulinum and C3stau2 from S. aureus - were crystallized. Here we present the 1.8 Å structure of C. limosum C3 transferase C3lim and compare it to the structures of other family members. In contrast to the structure of apo-C3bot, the canonical ADP-ribosylating turn turn motif is observed in a primed conformation, ready for NAD binding. This suggests an impact on the binding mode of NAD and on the transferase reaction. The crystal structure explains why auto-ADP-ribosylation of C3lim at Arg41 interferes with the ADP-ribosyltransferase activity of the toxin.     

Giardia duodenalis: Kinetics of cyst elimination and the systemic humoral and intestinal secretory immune responses in gerbils (Meriones unguiculatus) experimentally infected

This study aimed to determine the pre-patent period and to evaluate the kinetics of cyst elimination and the systemic humoral (IgA, IgG₁, IgG_2a, IgM, IgE) and intestinal secretory (IgA) immune responses in gerbils (Meriones unguiculatus) experimentally innoculated with different doses of Giardia duodenalis trophozoites. Forty-eight animals aged 6-8 weeks were used, equally distributed among six groups, five groups innoculated with different doses of trophozoites (10¹, 10², 10³, 10⁴, 10⁵) and one control (non-infected) group. Coproparasitological examinations were carried out daily up to 91 days after inoculation (d.a.i.) to determine the pre-patent period and the kinetics of cyst elimination. Blood and stool samples were weekly collected for antibody assays. The pre-patent period was observed from the 9 d.a.i. onwards, with intermittent elimination of variable quantities of cysts up to 27 d.a.i.. All infected gerbils, irrespective of the dose received, were able to mount systemic humoral immune responses as evidenced by specific IgM titers from 7 to 28 d.a.i., corresponding to the peak of cyst elimination, followed by high and persistent IgG1 titers. Intestinal secretory responses were also seen with two peaks of fecal IgA titers, corresponding to IgM and IgG1response peaks, respectively. In conclusion, systemic and intestinal humoral immune responses were related to the control of giardiasis in this experimental model.     

Plasmodium falciparum Genotype Diversity in Artemisinin Derivatives Treatment Failure Patients along the Thai-Myanmar Border

Genetic characteristics of Plasmodium falciparum may play a role in the treatment outcome of malaria infection. We have studied the association between diversity at the merozoite surface protein-1 (msp-1), msp-2, and glutamate-rich protein (glurp) loci and the treatment outcome of uncomplicated falciparum malaria patients along the Thai-Myanmar border who were treated with artemisinin derivatives combination therapy. P. falciparum isolates were collected prior to treatment from 3 groups of patients; 50 cases of treatment failures, 50 recrudescences, and 56 successful treatments. Genotyping of the 3 polymorphic markers was analyzed by nested PCR. The distribution of msp-1 alleles was significantly different among the 3 groups of patients but not the msp-2 and glurp alleles. The allelic frequencies of K1 and MAD20 alleles of msp1 gene were higher while RO33 allele was significantly lower in the successful treatment group. Treatment failure samples had a higher median number of alleles as compared to the successful treatment group. Specific genotypes of msp-1, msp-2, and glurp were significantly associated with the treatment outcomes. Three allelic size variants were significantly higher among the isolates from the treatment failure groups, i.e., K1_270-290, 3D7_610-630, G_650-690, while 2 variants, K1_150-170, and 3D7_670-690 were significantly lower. In conclusion, the present study reports the differences in multiplicity of infection and distribution of specific alleles of msp-1, msp-2, and glurp genes in P. falciparum isolates obtained from treatment failure and successful treatment patients following artemisinin derivatives combination therapy.     

Specificity and mutational analysis of the metal-dependent 3-deoxy-d-manno-octulosonate 8-phosphate synthase from Acidithiobacillus ferrooxidans

3-Deoxy-d-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the reaction between phosphoenol pyruvate and d-arabinose 5-phosphate to generate KDO8P. This reaction is part of the biosynthetic pathway to 3-deoxy-d-manno-octulosonate, a component of the lipopolysaccharide of the Gram-negative bacterial cell wall. Two distinct groups of KDO8PSs exist, differing by the absolute requirement of a divalent metal ion. In this study Acidithiobacillus ferrooxidans KDO8PS has been expressed and purified and shown to require a divalent metal ion, with Mn²⁺, Co²⁺ and Cd²⁺ (in decreasing order) being able to restore activity to metal-free enzyme. Cd²⁺ significantly enhanced the stability of the enzyme, raising the T_m by 14 °C. d-Glucose 6-phosphate and d-erythrose 4-phosphate were not substrates for A. ferrooxidans KDO8PS, whereas 2-deoxy-d-ribose 5-phosphate was a poor substrate and there was negligible activity with d-ribose 5-phosphate. The ²⁴³AspGlyPro²⁴⁵ motif is absolutely conserved in the metal-independent group of synthases, but the Gly and Pro sites are variable in the metal-dependent enzymes. Substitution of the putative metal-binding Asp243 to Ala in A. ferrooxidans KDO8PS gave inactive enzyme, whereas substitutions Asp243Glu or Pro245Ala produced active enzymes with altered metal-dependency profiles. Prior studies indicated that exchange of a metal-binding Cys for Asn converts metal-dependent KDO8P synthase into a metal-independent form. Unexpectedly, this mutation in A. ferrooxidans KDO8P synthase (Cys21Asn) gave inactive enzyme. This finding, together with modest activity towards 2-deoxy-d-ribose 5-phosphate suggests similarities between the A. ferrooxidans KDO8PS and the related metal-dependent 3-deoxy-d-arabino-heptulosonate phosphate synthase, and highlights the importance of the AspGlyPro loop in positioning the substrate for effective catalysis in all KDO8P synthases.     

Parameters determining the relative efficacy of hydroxy-naphthoquinone inhibitors of the cytochrome bc1 complex

Hydroxy-naphthoquinones are competitive inhibitors of the cytochrome bc₁ complex that bind to the ubiquinol oxidation site between cytochrome b and the iron-sulfur protein and presumably mimic a transition state in the ubiquinol oxidation reaction catalyzed by the enzyme. The parameters that affect efficacy of binding of these inhibitors to the bc₁ complex are not well understood. Atovaquone®, a hydroxy-naphthoquinone, has been used therapeutically to treat Pneumocystis carinii and Plasmodium infections. As the pathogens have developed resistance to this drug, it is important to understand the molecular basis of the drug resistance and to develop new drugs that can circumvent the drug resistance. We previously developed the yeast and bovine bc₁ complexes as surrogates to model the interaction of atovaquone with the bc₁ complexes of the target pathogens and human host. As a first step to identify new cytochrome bc₁ complex inhibitors with therapeutic potential and to better understand the determinants of inhibitor binding, we have screened a library of 2-hydroxy-naphthoquinones with aromatic, cyclic, and non-cyclic alkyl side-chain substitutions at carbon-3 on the hydroxy-quinone ring. We found a group of compounds with alkyl side-chains that effectively inhibit the yeast bc₁ complex. Molecular modeling of these into the crystal structure of the yeast cytochrome bc₁ complex provides structural and quantitative explanations for their binding efficacy to the target enzyme. In addition we also identified a 2-hydroxy-naphthoquinone with a branched side-chain that has potential for development as an anti-fungal and anti-parasitic therapeutic.