scholarly journals Missense Mutations in CytochromecMaturation Genes Provide New Insights into Rhodobacter capsulatus cbb3-Type CytochromecOxidase Biogenesis

2012 ◽  
Vol 195 (2) ◽  
pp. 261-269 ◽  
Author(s):  
Seda Ekici ◽  
Xinpei Jiang ◽  
Hans-Georg Koch ◽  
Fevzi Daldal
Keyword(s):  
Rhodobacter Capsulatus ◽  
Major Facilitator Superfamily ◽  
Missense Mutations ◽  
Content Type ◽  
Knockout Mutants ◽  
Major Facilitator ◽  
Copper Oxidase ◽  
First Time ◽  
Derivatives Of

ABSTRACTTheRhodobacter capsulatus cbb3-type cytochromecoxidase (cbb3-Cox) belongs to the heme-copper oxidase superfamily, and its subunits are encoded by theccoNOQPoperon. Biosynthesis of this enzyme is complex and needs dedicated biogenesis genes (ccoGHIS). It also relies on thec-type cytochrome maturation (Ccm) process, which requires theccmABCDEFGHIgenes, because two of thecbb3-Cox subunits (CcoO and CcoP) arec-type cytochromes. Recently, we reported that mutants lacking CcoA, a major facilitator superfamily type transporter, produce very small amounts ofcbb3-Cox unless the growth medium is supplemented with copper. In this work, we isolated “Cu-unresponsive” derivatives of accoAdeletion strain that exhibited nocbb3-Cox activity even upon Cu supplementation. Molecular characterization of these mutants revealed missense mutations in theccmAorccmFgene, required for the Ccm process. As expected, Cu-unresponsive mutants lacked the CcoO and CcoP subunits due to Ccm defects, but remarkably, they contained the CcoN subunit ofcbb3-Cox. Subsequent construction and examination of singleccmknockout mutants demonstrated that membrane insertion and stability of CcoN occurred in the absence of the Ccm process. Moreover, while theccmknockout mutants were completely incompetent for photosynthesis, the Cu-unresponsive mutants grew photosynthetically at lower rates and produced smaller amounts of cytochromesc1andc2than did a wild-type strain due to their restricted Ccm capabilities. These findings demonstrate that different levels of Ccm efficiency are required for the production of variousc-type cytochromes and reveal for the first time that maturation of the heme-Cu-containing subunit CcoN ofR. capsulatus cbb3-Cox proceeds independently of that of thec-type cytochromes during the biogenesis of this enzyme.

scholarly journals ThemelREDCAOperon Encodes a Utilization System for the Raffinose Family of Oligosaccharides inBacillus subtilis

2019 ◽  
Vol 201 (15) ◽  
Author(s):  
Kambiz Morabbi Heravi ◽  
Hildegard Watzlawick ◽  
Josef Altenbuchner
Keyword(s):  
Bacillus Subtilis ◽  
Transcriptional Start Site ◽  
Major Facilitator Superfamily ◽  
Atp Binding ◽  
Carbohydrate Utilization ◽  
Content Type ◽  
Storage Organs ◽  
Major Facilitator ◽  
Atp Binding Cassette

ABSTRACTBacillus subtilisis a heterotrophic soil bacterium that hydrolyzes different polysaccharides mainly found in the decomposed plants. These carbohydrates are mainly cellulose, hemicellulose, and the raffinose family of oligosaccharides (RFOs). RFOs are soluble α-galactosides, such as raffinose, stachyose, and verbascose, that rank second only after sucrose in abundance. Genome sequencing and transcriptome analysis ofB. subtilisindicated the presence of a putative α-galactosidase-encoding gene (melA) located in themsmRE-amyDC-melAoperon. Characterization of the MelA protein showed that it is a strictly Mn2+- and NAD+-dependent α-galactosidase able to hydrolyze melibiose, raffinose, and stachyose. Transcription of themsmER-amyDC-melAoperon is under control of a σA-type promoter located upstream ofmsmR(PmsmR), which is negatively regulated by MsmR. The activity of PmsmRwas induced in the presence of melibiose and raffinose. MsmR is a transcriptional repressor that binds to two binding sites at PmsmRlocated upstream of the −35 box and downstream of the transcriptional start site. MsmEX-AmyCD forms an ATP-binding cassette (ABC) transporter that probably transports melibiose into the cell. SincemsmRE-amyDC-melAis a melibiose utilization system, we renamed the operonmelREDCA.IMPORTANCEBacillus subtilisutilizes different polysaccharides produced by plants. These carbohydrates are primarily degraded by extracellular hydrolases, and the resulting oligo-, di-, and monosaccharides are transported into the cytosol via phosphoenolpyruvate-dependent phosphotransferase systems (PTS), major facilitator superfamily, and ATP-binding cassette (ABC) transporters. In this study, a new carbohydrate utilization system ofB. subtilisresponsible for the utilization of α-galactosides of the raffinose family of oligosaccharides (RFOs) was investigated. RFOs are synthesized from sucrose in plants and are mainly found in the storage organs of plant leaves. Our results revealed the modus operandi of a new carbohydrate utilization system inB. subtilis.

scholarly journals Uncovering the Transmembrane Metal Binding Site of the Novel Bacterial Major Facilitator Superfamily-Type Copper Importer CcoA

mBio ◽  
2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Bahia Khalfaoui-Hassani ◽  
Andreia F. Verissimo ◽  
Hans-Georg Koch ◽  
Fevzi Daldal
Keyword(s):  
Metal Binding ◽  
Rhodobacter Capsulatus ◽  
Bacterial Species ◽  
Major Facilitator Superfamily ◽  
Transmembrane Helices ◽  
Precise Control ◽  
Metal Ligands ◽  
Major Facilitator ◽  
Bacterial Cytoplasm ◽  
First Time

ABSTRACTUptake and trafficking of metals and their delivery to their respective metalloproteins are important processes. Cells need precise control of each step to avoid exposure to excessive metal concentrations and their harmful consequences. Copper (Cu) is a required micronutrient used as a cofactor in proteins. However, in large amounts, it can induce oxidative damage; hence, Cu homeostasis is indispensable for cell survival. Biogenesis of respiratory heme-Cu oxygen (HCO) reductases includes insertion of Cu into their catalytic subunits to form heme-Cu binuclear centers. Previously, we had shown that CcoA is a major facilitator superfamily (MFS)-type bacterial Cu importer required for biogenesis ofcbb3-type cytochromecoxidase (cbb3-Cox). Here, usingRhodobacter capsulatus, we focused on the import and delivery of Cu tocbb3-Cox. By comparing the CcoA amino acid sequence with its homologues from other bacterial species, we located several well-conserved Met, His, and Tyr residues that might be important for Cu transport. We determined the topology of the transmembrane helices that carry these residues to establish that they are membrane embedded, and substituted for them amino acids that do not ligand metal atoms. Characterization of these mutants for their uptake of radioactive64Cu andcbb3-Cox activities demonstrated that Met233 and His261 of CcoA are essential and Met237 and Met265 are important, whereas Tyr230 has no role for Cu uptake orcbb3-Cox biogenesis. These findings show for the first time that CcoA-mediated Cu import relies on conserved Met and His residues that could act as metal ligands at the membrane-embedded Cu binding domain of this transporter.IMPORTANCECu is a micronutrient that is both essential and toxic; hence, its cellular homeostasis is crucial. Respiratorycbb3-type cytochromecoxidases (cbb3-Cox) are Cu-containing energy-transducing enzymes that are important for many microaerophilic processes, including photosynthesis, respiration, and bacterial pathogenesis. How Cu is incorporated intocbb3-Cox enzymes is not well known. So far, CcoA is the only known major facilitator superfamily (MFS)-type transporter required for Cu import into the bacterial cytoplasm and forcbb3-Cox biogenesis. This study shows that the membrane-embedded, universally conserved Met and His residues of CcoA are essential for its Cu import function and also for its role incbb3-Cox biogenesis, shedding light on the mechanism of function of this bacterial prototypical Cu importer.

scholarly journals Intracytoplasmic Copper Homeostasis Controls Cytochrome c Oxidase Production

mBio ◽  
2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Seda Ekici ◽  
Serdar Turkarslan ◽  
Grzegorz Pawlik ◽  
Andrew Dancis ◽  
Nitin S. Baliga ◽  
...  
Keyword(s):  
Rhodobacter Capsulatus ◽  
Mitochondrial Diseases ◽  
Copper Homeostasis ◽  
Major Facilitator Superfamily ◽  
Content Type ◽  
Metal Cofactor ◽  
Respiratory Chains ◽  
Single Base Pair ◽  
Major Facilitator ◽  
Cellular Copper

ABSTRACTCopper is an essential micronutrient used as a metal cofactor by a variety of enzymes, including cytochromecoxidase (Cox). In all organisms from bacteria to humans, cellular availability and insertion of copper into target proteins are tightly controlled due to its toxicity. The major subunit of Cox contains a copper atom that is required for its catalytic activity. Previously, we identified CcoA (a member of major facilitator superfamily transporters) as a component required forcbb3-type Cox production in the Gram-negative, facultative phototrophRhodobacter capsulatus. Here, first we demonstrate that CcoA is a cytoplasmic copper importer. Second, we show that bypass suppressors of accoAdeletion mutant suppresscbb3-Cox deficiency by increasing cellular copper content and sensitivity. Third, we establish that these suppressors are single-base-pair insertion/deletions located incopA, encoding the major P1B-type ATP-dependent copper exporter (CopA) responsible for copper detoxification. AcopAdeletion alone has no effect oncbb3-Cox biogenesis in an otherwise wild-type background, even though it rescues thecbb3-Cox defect in the absence of CcoA and renders cells sensitive to copper. We conclude that a hitherto unknown functional interplay between the copper importer CcoA and the copper exporter CopA controls intracellular copper homeostasis required forcbb3-Cox production in bacteria likeR. capsulatus.IMPORTANCECopper (Cu) is an essential micronutrient required for many processes in the cell. It is found as a cofactor for heme-copper containing cytochromecoxidase enzymes at the terminus of the respiratory chains of aerobic organisms by catalyzing reduction of dioxygen (O2) to water. Defects in the biogenesis and copper insertion into cytochromecoxidases lead to mitochondrial diseases in humans. This work shows that a previously identified Cu transporter (CcoA) is a Cu importer and illustrates the link between two Cu transporters, the importer CcoA and the exporter CopA, required for Cu homeostasis and Cu trafficking to cytochromecoxidase in the cell.

scholarly journals Novel Transporter Required for Biogenesis of cbb3-Type Cytochrome c Oxidase in Rhodobacter capsulatus

mBio ◽  
2012 ◽  
Vol 3 (1) ◽  
Author(s):  
Seda Ekici ◽  
Honghui Yang ◽  
Hans-Georg Koch ◽  
Fevzi Daldal
Keyword(s):  
Rhodobacter Capsulatus ◽  
Crop Yields ◽  
Model Organism ◽  
Mitochondrial Diseases ◽  
Major Facilitator Superfamily ◽  
Content Type ◽  
Genomic Libraries ◽  
Cu Content ◽  
Major Facilitator ◽  
Cellular Copper

ABSTRACTThe acquisition, delivery, and incorporation of metals into their respective metalloproteins are important cellular processes. These processes are tightly controlled in order to prevent exposure of cells to free-metal concentrations that could yield oxidative damage. Copper (Cu) is one such metal that is required as a cofactor in a variety of proteins. However, when present in excessive amounts, Cu is toxic due to its oxidative capability. Cytochromecoxidases (Coxs) are among the metalloproteins whose assembly and activity require the presence of Cu in their catalytic subunits. In this study, we focused on the acquisition of Cu for incorporation into the heme-Cu binuclear center of thecbb3-type Cox (cbb3-Cox) in the facultative phototrophRhodobacter capsulatus. Genetic screens identified acbb3-Cox defective mutant that requires Cu2+supplementation to produce an activecbb3-Cox. Complementation of this mutant using wild-type genomic libraries unveiled a novel gene (ccoA) required forcbb3-Cox biogenesis. In the absence of CcoA, the cellular Cu content decreases andcbb3-Cox assembly and activity become defective. CcoA shows homology to major facilitator superfamily (MFS)-type transporter proteins. Members of this family are known to transport small solutes or drugs, but so far, no MFS protein has been implicated incbb3-Cox biogenesis. These findings provide novel insights into the maturation and assembly of membrane-integral metalloproteins and on a hitherto-unknown function(s) of MFS-type transporters in bacterial Cu acquisition.IMPORTANCEBiogenesis of energy-transducing membrane-integral enzymes, like the heme copper-containing cytochromecoxidases, and the acquisition of transition metals, like copper, as their catalytic cofactors are vital processes for all cells. These widespread and well-controlled processes are poorly understood in all organisms, including bacteria. Defects in these processes lead to severe mitochondrial diseases in humans and poor crop yields in plants. In this study, using the facultative phototrophRhodobacter capsulatusas a model organism, we report on the discovery of a novel major facilitator superfamily (MFS)-type transporter (CcoA) that affects cellular copper content andcbb3-type cytochromecoxidase production in bacteria.

scholarly journals DdvK, a Novel Major Facilitator Superfamily Transporter Essential for 5,5′-Dehydrodivanillate Uptake bySphingobiumsp. Strain SYK-6

2018 ◽  
Vol 84 (20) ◽  
Author(s):  
Kosuke Mori ◽  
Koh Niinuma ◽  
Masaya Fujita ◽  
Naofumi Kamimura ◽  
Eiji Masai
Keyword(s):  
Value Added ◽  
Major Facilitator Superfamily ◽  
Amino Acid Sequence Similarity ◽  
Aromatic Acids ◽  
Transporter Gene ◽  
Content Type ◽  
Biomass Resource ◽  
Major Facilitator ◽  
Mfs Transporter

ABSTRACTThe microbial conversion of lignin-derived aromatics is a promising strategy for the industrial utilization of this large biomass resource. However, efficient application requires an elucidation of the relevant transport and catabolic pathways. InSphingobiumsp. strain SYK-6, most of the enzyme genes involved in 5,5′-dehydrodivanillate (DDVA) catabolism have been characterized, but the transporter has not yet been identified. Here, we identified SLG_07710 (ddvK) and SLG_07780 (ddvR), genes encoding a putative major facilitator superfamily (MFS) transporter and MarR-type transcriptional regulator, respectively. AddvKmutant of SYK-6 completely lost the capacity to grow on and convert DDVA. DdvR repressed the expression of the DDVAO-demethylase oxygenase component gene (ligXa), while DDVA acted as the gene inducer. A DDVA uptake assay was developed by employing this DdvR-controlledligXatranscriptional regulatory system. ASphingobium japonicumUT26S transformant expressingddvKacquired DDVA uptake capacity, indicating thatddvKencodes the DDVA transporter. DdvK, probably requiring the proton motive force, was suggested to be a novel MFS transporter on the basis of the amino acid sequence similarity. Subsequently, we evaluated the effects ofddvKoverexpression on the production of the DDVA metabolite 2-pyrone-4,6-dicarboxylate (PDC), a building block of functional polymers. A SYK-6 mutant of the PDC hydrolase gene (ligI) cultured in DDVA accumulated PDC via 5-carboxyvanillate and grew by utilizing 4-carboxy-2-hydroxypenta-2,4-dienoate. The introduction of addvK-expression plasmid into aligImutant increased the growth rate in DDVA and the amounts of DDVA converted and PDC produced after 48 h by 1.35- and 1.34-fold, respectively. These results indicate that enhanced transporter gene expression can improve metabolite production from lignin derivatives.IMPORTANCEThe bioengineering of bacteria to selectively transport and metabolize natural substrates into specific metabolites is a valuable strategy for industrial-scale chemical production. The uptake of many substrates into cells requires specific transport systems, and so the identification and characterization of transporter genes are essential for industrial applications. A number of bacterial major facilitator superfamily transporters of aromatic acids have been identified and characterized, but many transporters of lignin-derived aromatic acids remain unidentified. The efficient conversion of lignin, an abundant but unutilized aromatic biomass resource, to value-added metabolites using microbial catabolism requires the characterization of transporters for lignin-derived aromatics. In this study, we identified the transporter gene responsible for the uptake of 5,5′-dehydrodivanillate, a lignin-derived biphenyl compound, inSphingobiumsp. strain SYK-6. In addition to characterizing its function, we applied this transporter gene to the production of a value-added metabolite from 5,5′-dehydrodivanillate.

scholarly journals Identification of pigments in artworks by inverse tangent derivative of spectrum and a new filtering method

2020 ◽  
Vol 8 (1) ◽  
Author(s):  
F. Fazlali ◽  
S. Gorji Kandi
Keyword(s):  
Reflectance Spectrum ◽  
Practical Method ◽  
Chemical Methods ◽  
Munk Function ◽  
Absorption Of Light ◽  
Spectrophotometric Data ◽  
First Time ◽  
Non Destructive ◽  
Derivatives Of

Abstract Employing an economical and non-destructive method for identifying pigments utilized in artworks is a significant aspect for preserving their antiquity value. One of the non-destructive methods for this purpose is spectrophotometry, which is based on the selected absorption of light. Mathematical descriptive methods such as derivatives of the reflectance spectrum, the Kubelka–Munk function and logarithm have been employed for the characterization of the peak features corresponding to the spectrophotometric data. In the present study, the mentioned mathematical descriptive methods were investigated with the aim to characterize the constituents of an Iranian artwork but were not efficient for the samples. Therefore, inverse tangent derivative equation was developed on spectral data for the first time, providing considerable details in the profile of reflectance curves. In the next part, to have a simpler and more practical method it was suggested to use filters made up of pure pigments. By using these filters and placing them on the samples, imaging was done. Then, images of samples with and without filter were evaluated and pure pigments were distinguished. The mentioned methods were also used to identify pigments in a modern Iranian painting specimen. The results confirmed these methods with reliable answers indicating that physical methods (alongside chemical methods) can also be effective in determining the types of pigments.

scholarly journals Two Different Quinohemoprotein Amine Dehydrogenases Initiate Anaerobic Degradation of Aromatic Amines inAromatoleum aromaticumEbN1

2019 ◽  
Vol 201 (16) ◽  
Author(s):  
Georg Schmitt ◽  
Martin Saft ◽  
Fabian Arndt ◽  
Jörg Kahnt ◽  
Johann Heider
Keyword(s):  
Aromatic Amines ◽  
Catalytic Efficiency ◽  
Growth Conditions ◽  
Complete Replacement ◽  
Content Type ◽  
Enzyme Family ◽  
First Time ◽  
Α Subunits ◽  
Substrate Spectrum

ABSTRACTAromatic amines like 2-phenylethylamine (2-PEA) and benzylamine (BAm) have been identified as novel growth substrates of the betaproteobacteriumAromatoleum aromaticumEbN1, which degrades a wide variety of aromatic compounds in the absence of oxygen under denitrifying growth conditions. The catabolic pathway of these amines was identified, starting with their oxidative deamination to the corresponding aldehydes, which are then further degraded via the enzymes of the phenylalanine or benzyl alcohol metabolic pathways. Two different periplasmic quinohemoprotein amine dehydrogenases involved in 2-PEA or BAm metabolism were identified and characterized. Both enzymes consist of three subunits, contain two hemeccofactors in their α-subunits, and exhibit extensive processing of their γ-subunits, generating four intramolecular thioether bonds and a cysteine tryptophylquinone (CTQ) cofactor. One of the enzymes was present in cells grown with 2-PEA or other substrates, showed an α2β2γ2composition, and had a rather broad substrate spectrum, which included 2-PEA, BAm, tyramine, and 1-butylamine. In contrast, the other enzyme was specifically induced in BAm-grown cells, showing an αβγ composition and activity only with BAm and 2-PEA. Since the former enzyme showed the highest catalytic efficiency with 2-PEA and the latter with BAm, they were designated 2-PEADH and benzylamine dehydrogenase (BAmDH). The catalytic properties and inhibition patterns of 2-PEADH and BAmDH showed considerable differences and were compared to previously characterized quinohemoproteins of the same enzyme family.IMPORTANCEThe known substrate spectrum ofA. aromaticumEbN1 is expanded toward aromatic amines, which are metabolized as sole substrates coupled to denitrification. The characterization of the two quinohemoprotein isoenzymes involved in degrading either 2-PEA or BAm expands the knowledge of this enzyme family and establishes for the first time that the necessary maturation of their quinoid CTQ cofactors does not require the presence of molecular oxygen. Moreover, the study revealed a highly interesting regulatory phenomenon, suggesting that growth with BAm leads to a complete replacement of 2-PEADH by BAmDH, which has considerably different catalytic and inhibition properties.

scholarly journals Construction and Application of a Functional Library of Cytochrome P450 Monooxygenases from the Filamentous Fungus Aspergillus oryzae

2011 ◽  
Vol 77 (9) ◽  
pp. 3147-3150 ◽  
Author(s):  
K. H. M. Nazmul Hussain Nazir ◽  
Hirofumi Ichinose ◽  
Hiroyuki Wariishi
Keyword(s):  
Cytochrome P450 ◽  
Aspergillus Oryzae ◽  
Cytochrome P450 Monooxygenases ◽  
Functional Screening ◽  
Content Type ◽  
P450 Monooxygenases ◽  
Rapid Characterization ◽  
Catalytic Functions ◽  
First Time

ABSTRACTA functional library of cytochrome P450 monooxygenases fromAspergillus oryzae(AoCYPs) was constructed in which 121 isoforms were coexpressed with yeast NADPH-cytochrome P450 oxidoreductase inSaccharomyces cerevisiae. Using this functional library, novel catalytic functions of AoCYPs, such as catalytic potentials of CYP57B3 against genistein, were elucidated for the first time. Comprehensive functional screening promises rapid characterization of catalytic potentials and utility of AoCYPs.

scholarly journals Fulminant Cryptosporidiosis after Near-Drowning: a Human Cryptosporidium parvum Strain Implicated in Invasive Gastrointestinal Adenocarcinoma and Cholangiocarcinoma in an Experimental Model

2012 ◽  
Vol 78 (6) ◽  
pp. 1746-1751 ◽  
Author(s):  
Gabriela Certad ◽  
Sadia Benamrouz ◽  
Karine Guyot ◽  
Anthony Mouray ◽  
Thierry Chassat ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cryptosporidium Parvum ◽  
Severe Infection ◽  
Digestive Cancer ◽  
Content Type ◽  
Near Drowning ◽  
Neoplastic Process ◽  
First Time

ABSTRACTIn the present work, we report the characterization of aCryptosporidium parvumstrain isolated from a patient who nearly drowned in the Deule River (Lille, France) after being discharged from the hospital where he had undergone allogeneic stem cell transplantation. After being rescued and readmitted to the hospital, he developed fulminant cryptosporidiosis. The strain isolated from the patient's stools was identified asC. parvumII2A15G2R1 (subtype linked to zoonotic exposure) and inoculated into SCID mice. In this host, this virulentC. parvumisolate induced not only severe infection but also invasive gastrointestinal and biliary adenocarcinoma. The observation of adenocarcinomas that progressed through all layers of the digestive tract to the subserosa and spread via blood vessels confirmed the invasive nature of the neoplastic process. These results indicate for the first time that a human-derivedC. parvumisolate is able to induce digestive cancer. This study is of special interest considering the exposure of a large number of humans and animals to this waterborne protozoan, which is highly tumorigenic when inoculated in a rodent model.

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