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Brian G Fox

Publications and source records attributed to Brian G Fox.

At least 19 recordsLinked to original sources
PubMed2006

Identification of Rv3230c as the NADPH oxidoreductase of a two-protein DesA3 acyl-CoA desaturase in Mycobacterium tuberculosis H37Rv.

DesA3 is a membrane-bound stearoyl-CoA Delta(9)-desaturase that produces oleic acid, a precursor of mycobacterial membrane phospholipids and triglycerides. The sequence of DesA3 is homologous with those of other membrane desaturases, including the presence of the eight-His motif proposed to bind the diiron center active site. This family of desaturases function as multicomponent complexes and thus require electron transfer proteins for efficient catalytic turnover. Here we present evidence that Rv3230c from Mycobacterium tuberculosis H37Rv is a biologically relevant electron transfer partner for DesA3 from the same pathogen. For these studies, Rv3230c was expressed as a partially soluble protein in Escherichia coli; recombinant DesA3 was expressed in Mycobacterium smegmatis as a catalytically active membrane protein. The addition of E. coli lysates containing Rv3230c to lysates of M. smegmatis expressing DesA3 gave strong conversion of [1-(14)C]-18:0-CoA to [1-(14)C]-cis-Delta(9)-18:1-CoA and of [1-(14)C]-16:0-CoA to [1-(14)C]-cis-Delta(9)-16:1-CoA. Both M. tuberculosis proteins were required for reconstitution of activity, as various combinations of control lysates lacking either Rv3230c or DesA3 gave minimal or no activity. Furthermore, the specificity of interaction between Rv3230c and DesA3 was implied by the inability of other related redox systems to substitute for Rv3230c. The reconstituted activity was dependent upon the presence of NADPH, could be saturated by increasing the amount of Rv3230c added, and was also sensitive to the salt concentration in the buffer. The results are consistent with the formation of a protein-protein complex, possibly with electrostatic character. This work defines a multiprotein, acyl-CoA desaturase complex from M. tuberculosis H37Rv to minimally consist of a soluble Rv3230c reductase and integral membrane DesA3 desaturase. Further implications of this finding relative to the properties of other multiprotein iron-enzyme complexes are discussed.

Amino Acid Sequence↗
PubMed2006

Redox reactions of dehydroepiandrosterone and its metabolites in differentiating 3T3-L1 adipocytes: A liquid chromatographic-mass spectrometric study.

Dehydroepiandrosterone is known to depress fatty acid formation in differentiating 3T3-L1 adipocytes. The metabolism of dehydroepiandrosterone and four of its natural metabolites in differentiating adipocytes was studied by liquid chromatography-mass spectrometry. Adipocytes rapidly converted dehydroepiandrosterone to androst-5-ene-3beta,17beta-diol. 7alpha-Hydroxy-DHEA was interconverted with 7-oxo-DHEA and 7beta-hydroxy-DHEA and the corresponding 17beta reduced products. Dehydroepiandrosterone and its derivatives were detected only in the culture medium suggesting that dehydroepiandrosterone is metabolized via enzymes located in close proximity to, or that are integral parts of the cell membrane. Alternatively, there may be efficient mechanisms at play for extrusion of the steroids to the aqueous media rather than being retained in the lipid-rich cell. An interesting aspect of the study was finding androstenediol as the major metabolite of dehydroepiandrosterone. Androst-5-ene-3beta,17beta-diol has been implicated in prostate cancer. The contribution of adipose cells to the circulating supply of androst-5-ene-3beta,17beta-diol may therefore be considered in managing prostate cancer.

3T3-L1 Cells↗
PubMed2006

Component interactions and implications for complex formation in the multicomponent toluene 4-monooxygenase.

A fluorophore-labeled form of the T4moD, the catalytic effector protein of the toluene 4-monooxygenase complex, was prepared by engineering the N-terminal region to contain a tetraCys motif and treatment with biarsenical fluorescein. Fluorescence anisotropy was used to study the protein-protein interactions among various combinations of the four components of the complex. Binding interactions were detected between T4moD and the hydroxylase component T4moH [K(D) value of 83 nM for interaction with the alphabetagamma protomer] and between T4moD and the Rieske [2Fe-2S] ferredoxin component T4moC (K(D) value of 78 nM). No binding interactions were detected between T4moD and the NADH oxidoreductase component T4moF, but T4moF was able to disrupt binding between T4moC and T4moD. The detected binding interactions suggest an intermediary electron transfer complex between T4moC and T4moD that excludes T4moF. The results indicate that specialization of effector protein function may include specific protein-protein interactions with [2Fe-2S] domains as well as the hydroxylase component.

Arsenicals↗
PubMed2006

Solution structures of spinach acyl carrier protein with decanoate and stearate.

Acyl carrier protein (ACP) is a cofactor in a variety of biosynthetic pathways, including fatty acid metabolism. Thus, it is of interest to determine structures of physiologically relevant ACP-fatty acid complexes. We report here the NMR solution structures of spinach ACP with decanoate (10:0-ACP) and stearate (18:0-ACP) attached to the 4'-phosphopantetheine prosthetic group. The protein in the fatty acid complexes adopts a single conformer, unlike apo- and holo-ACP, which interconvert in solution between two major conformers. The protein component of both 10:0- and 18:0-ACP adopts the four-helix bundle topology characteristic of ACP, and a fatty acid binding cavity was identified in both structures. Portions of the protein close in space to the fatty acid and the 4'-phosphopantetheine were identified using filtered/edited NOESY experiments. A docking protocol was used to generate protein structures containing bound fatty acid for 10:0- and 18:0-ACP. In both cases, the predominant structure contained fatty acid bound down the center of the helical bundle, in agreement with the location of the fatty acid binding pockets. These structures demonstrate the conformational flexibility of spinach ACP and suggest how the protein changes to accommodate its myriad binding partners.

Acyl Carrier Protein↗
PubMed2006

Structure of T4moC, the Rieske-type ferredoxin component of toluene 4-monooxygenase.

The structure of the Rieske-type ferredoxin (T4moC) from toluene 4-monooxygenase was determined by X-ray crystallography in the [2Fe-2S](2+) state at a resolution of 1.48 A using single-wavelength anomalous dispersion phasing with the [2Fe-2S] center. The structure consists of ten beta-strands arranged into the three antiparallel beta-sheet topology observed in all Rieske proteins. Trp69 of T4moC is adjacent to the [2Fe-2S] centre, which displaces a loop containing the conserved Pro81 by approximately 8 A away from the [2Fe-2S] cluster compared with the Pro loop in the closest structural and functional homolog, the Rieske-type ferredoxin BphF from biphenyl dioxygenase. In addition, T4moC contains five hydrogen bonds to the [2Fe-2S] cluster compared with three hydrogen bonds in BphF. Moreover, the electrostatic surface of T4moC is distinct from that of BphF. These structural differences are identified as possible contributors to the evolutionary specialization of soluble Rieske-type ferredoxins between the diiron monooxygenases and cis-dihydrodiol-forming dioxygenases.

Crystallography, X-Ray↗
PubMed2006

Identification of the binding region of the [2Fe-2S] ferredoxin in stearoyl-acyl carrier protein desaturase: insight into the catalytic complex and mechanism of action.

Stearoyl-acyl carrier protein desaturase (Delta9D) catalyzes the O(2) and 2e(-) dependent desaturation of stearoyl-acyl carrier protein (18:0-ACP) to yield oleoyl-ACP (18:1-ACP). The 2e(-) are provided by essential interactions with reduced plant-type [2Fe-2S] ferredoxin (Fd). We have investigated the protein-protein interface involved in the Fd-Delta9D complex by the use of chemical cross-linking, site-directed mutagenesis, steady-state kinetic approaches, and molecular docking studies. The treatment of the different proteins with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide revealed that carboxylate residues from Fd and lysine residues from Delta9D contribute to cross-linking. The single substitutions of K60A, K56A, and K230A on Delta9D decreased the k(cat)/K(M) for Fd by 4-, 22-, and 2400-fold, respectively, as compared to wt Delta9D and a K41A substitution. The double substitution K56A/K60A decreased the k(cat)/K(M) for Fd by 250-fold, whereas the triple mutation K56A/K60A/K230A decreased the k(cat)/K(M) for Fd by at least 700 000-fold. These results strongly implicate the triad of K56, K60, and K230 of Delta9D in the formation of a catalytic complex with Fd. Molecular docking studies indicate that electrostatic interactions between K56 and K60 and the carboxylate groups on Fd may situate the [2Fe-2S] cluster of Fd closer to W62, a surface residue that is structurally conserved in both ribonucleotide reductase and mycobacterial putative acyl-ACP desaturase DesA2. Owing to the considerably larger effects on catalysis, K230 appears to have other contributions to catalysis arising from its positioning in helix 7 and its close spatial location to the diiron center ligands E229 and H232. These results are considered in the light of the presently available models for Fd-mediated electron transfer in Delta9D and other protein-protein complexes.

Binding Sites↗
PubMed2006

Structure and mechanism of mouse cysteine dioxygenase.

Cysteine dioxygenase (CDO) catalyzes the oxidation of l-cysteine to cysteine sulfinic acid. Deficiencies in this enzyme have been linked to autoimmune diseases and neurological disorders. The x-ray crystal structure of CDO from Mus musculus was solved to a nominal resolution of 1.75 Angstroms. The sequence is 91% identical to that of a human homolog. The structure reveals that CDO adopts the typical beta-barrel fold of the cupin superfamily. The NE2 atoms of His-86, -88, and -140 provide the metal binding site. The structure further revealed a covalent linkage between the side chains of Cys-93 and Tyr-157, the cysteine of which is conserved only in eukaryotic proteins. Metal analysis showed that the recombinant enzyme contained a mixture of iron, nickel, and zinc, with increased iron content associated with increased catalytic activity. Details of the predicted active site are used to present and discuss a plausible mechanism of action for the enzyme.

Amino Acid Sequence↗
PubMed2005

High efficiency single step production of expression plasmids from cDNA clones using the Flexi Vector cloning system.

The success of structural genomics and proteomics initiatives is dependent on the availability of target genes in vectors suitable for protein production. Here, we compare two high-throughput methods for producing expression vectors from plasmid-derived cDNA fragments. Expression vectors were constructed for compatibility with the Gateway recombination cloning system and the Flexi Vector restriction-based cloning system. Cloning protocols for each system were conducted in parallel for 96 different target genes from PCR through the production of sequence-verified expression clones. The short nucleotide sequences required to prepare the target open reading frames for Flexi Vector cloning allowed a single-step PCR protocol, resulting in fewer mutations relative to the Gateway protocol. Furthermore, through initial cloning of the target open reading frames directly into an expression vector, the Flexi Vector system gave time and cost savings compared to the protocol required for the Gateway system. Within the Flexi Vector system, genes were transferred between four different expression vectors. The efficiency of gene transfer between Flexi Vectors depended on including a region of sequence identity adjacent to one of the restriction sites. With the proper construction in the flanking sequence of the vector, gene transfer efficiencies of 95-98% were demonstrated.

Cloning, Molecular↗
PubMed2005

Preparation of isotopically labeled spinach acyl-acyl carrier protein for NMR structural studies.

Acyl carrier proteins (ACPs) are important protein cofactors in fatty acid biosynthesis, but their acylated forms have not been well-studied. To permit detailed nuclear magnetic resonance studies of acylated spinach ACP isoform I, we have developed a new expression plasmid for recombinant production of the apo-protein and modified protocols for purifying the protein product and acylating it to form acyl-ACP. To solve plasmid stability problems associated with growth in minimal media, the ampicillin resistance gene from pSACP-2a was replaced with the tetA(C) gene from pBR322. The resulting plasmid, pSACP-2t, supported overexpression of apo-ACP in Escherichia coli BL21(DE3) cells in M9 medium containing 15NH4Cl as the sole nitrogen source. Apo-ACP was purified to homogeneity by means of polyethylene glycol precipitation and anion exchange. Two in vitro synthetic routes were used to produce acyl-ACPs. In one route, apo-ACP was converted to the holo form and the acyl form by a published protocol that employs a discrete enzymatic reaction for each step. As an alternative route to produce decanoyl-ACP, apo-ACP was directly converted to the acyl form by using holo-ACP synthase along with the non-natural substrate decanoyl-CoA. Two-dimensional 1H-15N NMR spectroscopy of decanoyl-ACP and stearoyl-ACP revealed that changes in the length of the covalently attached fatty acid do not affect the secondary structure of the protein but do influence the local conformation and dynamics.

Acyl Carrier Protein↗
PubMed2005

Oxygen-18 tracer studies of enzyme reactions with radical/cation diagnostic probes.

This review considers reactions of enzymes with the cyclopropanoid radical/cation diagnostic probes norcarane, 1,1-dimethylcyclopropane, and 1,1-diethylcyclopropane as elaborated by the use of 18O2 and 18OH2 to trace the origin of O-atoms incorporated during catalysis. The reactions of soluble and integral membrane diiron enzymes are summarized and compared to results obtained from cytochrome P450 studies. Norcarane proved to be an excellent substrate for the diiron enzyme toluene 4-monooxygenase and its engineered isoforms, with kcat and coupling between NADH utilization and total hydroxylated products comparable to that determined for toluene, the natural substrate. Results obtained with toluene 4-monooxygenase show that the un-rearranged and radical-rearranged alcohol products have a high percentage of O-atom incorporation (>80-95%) from O2, while the cation-derived ring-expansion products have O-atom incorporation primarily derived from solvent water. Mechanistic possibilities accounting for this difference are discussed.

Cations↗
PubMed2005

Crystal structures and functional studies of T4moD, the toluene 4-monooxygenase catalytic effector protein.

Toluene 4-monooxygenase (T4MO) is a four-component complex that catalyzes the regiospecific, NADH-dependent hydroxylation of toluene to yield p-cresol. The catalytic effector (T4moD) of this complex is a 102-residue protein devoid of metals or organic cofactors. It forms a complex with the diiron hydroxylase component (T4moH) that influences both the kinetics and regiospecificity of catalysis. Here, we report crystal structures for native T4moD and two engineered variants with either four (DeltaN4-) or 10 (DeltaN10-) residues removed from the N-terminal at 2.1-, 1.7-, and 1.9-A resolution, respectively. The crystal structures have C-alpha root-mean-squared differences of less than 0.8 A for the central core consisting of residues 11-98, showing that alterations of the N-terminal have little influence on the folded core of the protein. The central core has the same fold topology as observed in the NMR structures of T4moD, the methane monooxygenase effector protein (MmoB) from two methanotrophs, and the phenol hydroxylase effector protein (DmpM). However, the root-mean-squared differences between comparable C-alpha positions in the X-ray structures and the NMR structures vary from approximately 1.8 A to greater than 6 A. The X-ray structures exhibit an estimated overall coordinate error from 0.095 (0.094) A based on the R-value (R free) for the highest resolution DeltaN4-T4moD structure to 0.211 (0.196) A for the native T4moD structure. Catalytic studies of the DeltaN4-, DeltaN7-, and DeltaN10- variants of T4moD show statistically insignificant changes in k(cat), K(M), k(cat)/K(M), and K(I) relative to the native protein. Moreover, there was no significant change in the regiospecificity of toluene oxidation with any of the T4moD variants. The relative insensitivity to changes in the N-terminal region distinguishes T4moD from the MmoB homologues, which each require the approximately 33 residue N-terminal region for catalytic activity.

Amino Acid Sequence↗
PubMed2005

Comparison of cell-based and cell-free protocols for producing target proteins from the Arabidopsis thaliana genome for structural studies.

We describe a comparative study of protein production from 96 Arabidopsis thaliana open reading frames (ORFs) by cell-based and cell-free protocols. Each target was carried through four pipeline protocols used by the Center for Eukaryotic Structural Genomics (CESG), one for the production of unlabeled protein to be used in crystallization trials and three for the production of 15N-labeled proteins to be analyzed by 1H-15N NMR correlation spectroscopy. Two of the protocols involved Escherichia coli cell-based and two involved wheat germ cell-free technology. The progress of each target through each of the protocols was followed with all failures and successes noted. Failures were of the following types: ORF not cloned, protein not expressed, low protein yield, no cleavage of fusion protein, insoluble protein, protein not purified, NMR sample too dilute. Those targets that reached the goal of analysis by 1H-15N NMR correlation spectroscopy were scored as HSQC+ (protein folded and suitable for NMR structural analysis), HSQC+/- (protein partially disordered or not in a single stable conformational state), HSQC- (protein unfolded, misfolded, or aggregated and thus unsuitable for NMR structural analysis). Targets were also scored as X- for failing to crystallize and X+ for successful crystallization. The results constitute a rich database for understanding differences between targets and protocols. In general, the wheat germ cell-free platform offers the advantage of greater genome coverage for NMR-based structural proteomics whereas the E. coli platform when successful yields more protein, as currently needed for crystallization trials for X-ray structure determination.

Arabidopsis↗
PubMed2005

The structure at 2.4 A resolution of the protein from gene locus At3g21360, a putative Fe(II)/2-oxoglutarate-dependent enzyme from Arabidopsis thaliana.

The crystal structure of the gene product of At3g21360 from Arabidopsis thaliana was determined by the single-wavelength anomalous dispersion method and refined to an R factor of 19.3% (Rfree = 24.1%) at 2.4 A resolution. The crystal structure includes two monomers in the asymmetric unit that differ in the conformation of a flexible domain that spans residues 178-230. The crystal structure confirmed that At3g21360 encodes a protein belonging to the clavaminate synthase-like superfamily of iron(II) and 2-oxoglutarate-dependent enzymes. The metal-binding site was defined and is similar to the iron(II) binding sites found in other members of the superfamily.

Arabidopsis↗
PubMed2005

Identification of transcribed sequences in Arabidopsis thaliana by using high-resolution genome tiling arrays.

Using a maskless photolithography method, we produced DNA oligonucleotide microarrays with probe sequences tiled throughout the genome of the plant Arabidopsis thaliana. RNA expression was determined for the complete nuclear, mitochondrial, and chloroplast genomes by tiling 5 million 36-mer probes. These probes were hybridized to labeled mRNA isolated from liquid grown T87 cells, an undifferentiated Arabidopsis cell culture line. Transcripts were detected from at least 60% of the nearly 26,330 annotated genes, which included 151 predicted genes that were not identified previously by a similar genome-wide hybridization study on four different cell lines. In comparison with previously published results with 25-mer tiling arrays produced by chromium masking-based photolithography technique, 36-mer oligonucleotide probes were found to be more useful in identifying intron-exon boundaries. Using two-dimensional HPLC tandem mass spectrometry, a small-scale proteomic analysis was performed with the same cells. A large amount of strongly hybridizing RNA was found in regions "antisense" to known genes. Similarity of antisense activities between the 25-mer and 36-mer data sets suggests that it is a reproducible and inherent property of the experiments. Transcription activities were also detected for many of the intergenic regions and the small RNAs, including tRNA, small nuclear RNA, small nucleolar RNA, and microRNA. Expression of tRNAs correlates with genome-wide amino acid usage.

Arabidopsis↗
PubMed2005

Results from high-throughput DNA cloning of Arabidopsis thaliana target genes using site-specific recombination.

The Center for Eukaryotic Structural Genomics (CESG) was founded as a collaborative effort to develop technologies for the rapid and economic determination of protein three-dimensional structures. The initial focus was on the genome of the model plant Arabidopsis thaliana. Protocols for high-throughput cloning of Arabidopsis open reading frames into Escherichia coli expression vectors are presented along with an analysis of results from approximately 2000 cloning experiments. Open reading frames were chosen on the likelihood that they would represent important unknown regions of protein conformation and fold space or that they would elucidate novel fold-function relationships. The chosen open reading frames were amplified from a cDNA pool created by reverse transcription of RNA isolated from an Arabidopsis callus culture. A novel Gateway protocol was developed to insert the amplified open reading frames into an entry vector for storage and sequence determination. Sequence verified entry clones were then used to create expression vectors again via the Gateway system.

Arabidopsis↗
PubMed2005

Sulfate requirement for heterotrophic growth of "Ferroplasma acidarmanus" strain fer1.

Growth of the acidophilic archaeon, "Ferroplasma acidarmanus" strain fer1, in a laboratory medium (primary constituents, FeSO4 72 mM and 0.02% yeast extract) is minimal. A survey of the annotated genome revealed metabolic transporters for Ni2+, sugars, and amino acids. Accordingly, the concentration of yeast extract was increased to 0.1% and the addition of 2 mM Ni(NH4)2(SO4)2 significantly enhanced the cultivation of strain fer1. The maximum optical density in the modified fer1 medium (mfer) was OD(492)=0.27 with 10(10) viable cells/ml as determined by a most-probable-number method, which exceeds previously reported viable cells/ml by >100-fold. Strain fer1 displayed chemolithotrophic growth with Fe2+ in mfer containing 100 mM FeSO4 or FeCl2. In the absence of Fe2+, heterotrophic growth occurred with one of the following salts (100 mM): ZnSO4, MnSO4, MgSO4, (NH4)2SO4, or Fe2(SO4)3, and did not occur with (100 mM): ZnCl2, MnCl2, MgCl2, NH4Cl, or FeCl3. Escaping headspace gas from strain fer1 cultures formed a precipitate in a zinc acetate trap. Sulfide was absent in the precipitate but zinc and sulfur were detected. These data demonstrate that SO4 is required for heterotrophic growth of strain fer1 and may have a role in the global sulfur cycle.

Archaea↗