Pleiotropic functions of a Streptomyces pristinaespiralis autoregulator receptor in development, antibiotic biosynthesis, and expression of a superoxide dismutase

In Streptomyces, a family of related butyrolactones and their corresponding receptor proteins serve as quorum-sensing systems that can activate morphological development and antibiotic biosynthesis. Streptomyces pristinaespiralis contains a gene cluster encoding enzymes and regulatory proteins for the biosynthesis of pristinamycin, a clinically important streptogramin antibiotic complex. One of these proteins, PapR1, belongs to a well known family of Streptomyces antibiotic regulatory proteins. Gel shift assays using crude cytoplasmic extracts detected SpbR, a developmentally regulated protein that bound to the papR1 promoter. SpbR was purified, and its gene was cloned using reverse genetics. spbR encoded a 25-kDa protein similar to Streptomyces autoregulatory proteins of the butyrolactone receptor family, including scbR from Streptomyces coelicolor. In Escherichia coli, purified SpbR and ScbR produced bound sequences immediately upstream of papR1, spbR, and scbR. SpbR DNA-binding activity was inhibited by an extracellular metabolite with chromatographic properties similar to those of the well known gamma-butyrolactone signaling compounds. DNase I protection assays mapped the SpbR-binding site in the papR1 promoter to a sequence homologous to other known butyrolactone autoregulatory elements. A nucleotide data base search showed that these binding motifs were primarily located upstream of genes encoding Streptomyces antibiotic regulatory proteins and butyrolactone receptors in various Streptomyces species. Disruption of the spbR gene in S. pristinaespiralis resulted in severe defects in growth, morphological differentiation, pristinamycin biosynthesis, and expression of a secreted superoxide dismutase.     

Microbial Reduction of 1,3-Dioxo-2-Methyl-2-(3′-Oxo-6′-Carbomethoxyhexyl)-Cyclopentane to Form 1β-Hydroxy-3-Oxo-2β-Methyl-2α-(3′-Oxo-6′-Carbomethoxy-hexyl)-Cyclopentane, an Intermediate for Steroid Total Synthesis

The rate and extent of stereoselective reduction of 1,3-dioxo-2-methyl-2-(3′-oxo-6′-carbomethoxyhexyl)-cyclopentane to form the 1β-hydroxy-2β-methyl isomer by cultures of Schizosaccharomyces pombe ATCC 2476 was dramatically increased by addition to the fermentation of certain α,β-unsaturated ketones and allyl alcohol.     

Occurrence of a putative ancient-like isomerase involved in histidine and tryptophan biosynthesis

We report the occurrence of an isomerase with a putative (βα)₈-barrel structure involved in both histidine and trypto-phan biosynthesis in Streptomyces coelicolor A3(2) and Mycobacterium tuberculosis HR37Rv. Deletion of a hisA homologue (SCO2050) putatively encoding N′-[(5′-phosphoribosyl)-formimino]-5 amino-imidazole-4-carboxamide ribonucleotide isomerase from the chromosome of S. coelicolor A3(2) generated a double auxotrophic mutant for histidine and tryptophan. The bifunctional gene SCO2050 and its orthologue Rv1603 from M. tuberculosis complemented both hisA and trpF mutants of Escherichia coli. Expression of the E. coli trpF gene in the S. coelicolor mutant only complemented the tryptophan auxo-trophy, and the hisA gene only complemented the histidine auxotrophy. The discovery of this enzyme, which has a broad-substrate specificity, has implications for the evolution of metabolic pathways and may prove to be important for understanding the evolution of the (βα)₈-barrels.     

Aprataxin, causative gene product for EAOH/AOA1, repairs DNA single-strand breaks with damaged 3'-phosphate and 3'-phosphoglycolate ends

Aprataxin is the causative gene product for early-onset ataxia with ocular motor apraxia and hypoalbuminemia/ataxia with oculomotor apraxia type 1 (EAOH/AOA1), the clinical symptoms of which are predominantly neurological. Although aprataxin has been suggested to be related to DNA single-strand break repair (SSBR), the physiological function of aprataxin remains to be elucidated. DNA single-strand breaks (SSBs) continually produced by endogenous reactive oxygen species or exogenous genotoxic agents, typically possess damaged 3′-ends including 3′-phosphate, 3′-phosphoglycolate, or 3′-α, β-unsaturated aldehyde ends. These damaged 3′-ends should be restored to 3′-hydroxyl ends for subsequent repair processes. Here we demonstrate by in vitro assay that recombinant human aprataxin specifically removes 3′-phosphoglycolate and 3′-phosphate ends at DNA 3′-ends, but not 3′-α, β-unsaturated aldehyde ends, and can act with DNA polymerase β and DNA ligase III to repair SSBs with these damaged 3′-ends. Furthermore, disease-associated mutant forms of aprataxin lack this removal activity. The findings indicate that aprataxin has an important role in SSBR, that is, it removes blocking molecules from 3′-ends, and that the accumulation of unrepaired SSBs with damaged 3′-ends underlies the pathogenesis of EAOH/AOA1. The findings will provide new insight into the mechanism underlying degeneration and DNA repair in neurons.     

Galactosyl-Lactose Sialylation Using Trypanosoma cruzi trans-Sialidase as the Biocatalyst and Bovine κ-Casein-Derived Glycomacropeptide as the Donor Substrate

trans-Sialidase (TS) enzymes catalyze the transfer of sialyl (Sia) residues from Sia(α2-3)Gal(β1-x)-glycans (sialo-glycans) to Gal(β1-x)-glycans (asialo-glycans). Aiming to apply this concept for the sialylation of linear and branched (Gal)_nGlc oligosaccharide mixtures (GOS) using bovine κ-casein-derived glycomacropeptide (GMP) as the sialic acid donor, a kinetic study has been carried out with three components of GOS, i.e., 3′-galactosyl-lactose (β3′-GL), 4′-galactosyl-lactose (β4′-GL), and 6′-galactosyl-lactose (β6′-GL). This prebiotic GOS is prepared from lactose by incubation with suitable β-galactosidases, whereas GMP is a side-stream product of the dairy industry. The trans-sialidase from Trypanosoma cruzi (TcTS) was expressed in Escherichia coli and purified. Its temperature and pH optima were determined to be 25°C and pH 5.0, respectively. GMP [sialic acid content, 3.6% (wt/wt); N-acetylneuraminic acid (Neu5Ac), >99%; (α2-3)-linked Neu5Ac, 59%] was found to be an efficient sialyl donor, and up to 95% of the (α2-3)-linked Neu5Ac could be transferred to lactose when a 10-fold excess of this acceptor substrate was used. The products of the TcTS-catalyzed sialylation of β3′-GL, β4′-GL, and β6′-GL, using GMP as the sialic acid donor, were purified, and their structures were elucidated by nuclear magnetic resonance spectroscopy. Monosialylated β3′-GL and β4′-GL contained Neu5Ac connected to the terminal Gal residue; however, in the case of β6′-GL, TcTS was shown to sialylate the 3 position of both the internal and terminal Gal moieties, yielding two different monosialylated products and a disialylated structure. Kinetic analyses showed that TcTS had higher affinity for the GL substrates than lactose, while the V_max and k_cat values were higher in the case of lactose.     

Identification of Propofol Binding Sites in a Nicotinic Acetylcholine Receptor with a Photoreactive Propofol Analog

Propofol, a widely used intravenous general anesthetic, acts at anesthetic concentrations as a positive allosteric modulator of γ-aminobutyric acid type A receptors and at higher concentration as an inhibitor of nicotinic acetylcholine receptors (nAChRs). Here, we characterize propofol binding sites in a muscle-type nAChR by use of a photoreactive analog of propofol, 2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol (AziPm). Based upon radioligand binding assays, AziPm stabilized the Torpedo nAChR in the resting state, whereas propofol stabilized the desensitized state. nAChR-rich membranes were photolabeled with [³H]AziPm, and labeled amino acids were identified by Edman degradation. [³H]AziPm binds at three sites within the nAChR transmembrane domain: (i) an intrasubunit site in the δ subunit helix bundle, photolabeling in the nAChR desensitized state (+agonist) δM2-18′ and two residues in δM1 (δPhe-232 and δCys-236); (ii) in the ion channel, photolabeling in the nAChR resting, closed channel state (−agonist) amino acids in the M2 helices (αM2-6′, βM2-6′ and -13′, and δM2-13′) that line the channel lumen (with photolabeling reduced by >90% in the desensitized state); and (iii) at the γ-α interface, photolabeling αM2-10′. Propofol enhanced [³H]AziPm photolabeling at αM2-10′. Propofol inhibited [³H]AziPm photolabeling within the δ subunit helix bundle at lower concentrations (IC₅₀ = 40 μm) than it inhibited ion channel photolabeling (IC₅₀ = 125 μm). These results identify for the first time a single intrasubunit propofol binding site in the nAChR transmembrane domain and suggest that this is the functionally relevant inhibitory binding site.     

Engineered and Native Coenzyme B12-dependent Isovaleryl-CoA/Pivalyl-CoA Mutase

Adenosylcobalamin-dependent isomerases catalyze carbon skeleton rearrangements using radical chemistry. We have recently demonstrated that an isobutyryl-CoA mutase variant, IcmF, a member of this enzyme family that catalyzes the interconversion of isobutyryl-CoA and n-butyryl-CoA also catalyzes the interconversion between isovaleryl-CoA and pivalyl-CoA, albeit with low efficiency and high susceptibility to inactivation. Given the biotechnological potential of the isovaleryl-CoA/pivalyl-CoA mutase (PCM) reaction, we initially attempted to engineer IcmF to be a more proficient PCM by targeting two active site residues predicted based on sequence alignments and crystal structures, to be key to substrate selectivity. Of the eight mutants tested, the F598A mutation was the most robust, resulting in an ∼17-fold increase in the catalytic efficiency of the PCM activity and a concomitant ∼240-fold decrease in the isobutyryl-CoA mutase activity compared with wild-type IcmF. Hence, mutation of a single residue in IcmF tuned substrate specificity yielding an ∼4000-fold increase in the specificity for an unnatural substrate. However, the F598A mutant was even more susceptible to inactivation than wild-type IcmF. To circumvent this limitation, we used bioinformatics analysis to identify an authentic PCM in genomic databases. Cloning and expression of the putative AdoCbl-dependent PCM with an α₂β₂ heterotetrameric organization similar to that of isobutyryl-CoA mutase and a recently characterized archaeal methylmalonyl-CoA mutase, allowed demonstration of its robust PCM activity. To simplify kinetic analysis and handling, a variant PCM-F was generated in which the αβ subunits were fused into a single polypeptide via a short 11-amino acid linker. The fusion protein, PCM-F, retained high PCM activity and like PCM, was resistant to inactivation. Neither PCM nor PCM-F displayed detectable isobutyryl-CoA mutase activity, demonstrating that PCM represents a novel 5′-deoxyadenosylcobalamin-dependent acyl-CoA mutase. The newly discovered PCM and the derivative PCM-F, have potential applications in bioremediation of pivalic acid found in sludge, in stereospecific synthesis of C5 carboxylic acids and alcohols, and in the production of potential commodity and specialty chemicals.     

The Biosynthetic pathway for synechoxanthin, an aromatic carotenoid synthesized by the euryhaline, unicellular cyanobacterium Synechococcus sp. strain PCC 7002

The euryhaline, unicellular cyanobacterium Synechococcus sp. strain PCC 7002 produces the dicyclic aromatic carotenoid synechoxanthin (χ,χ-caroten-18,18′-dioic acid) as a major pigment (>15% of total carotenoid) and when grown to stationary phase also accumulates small amounts of renierapurpurin (χ,χ-carotene) (J. E. Graham, J. T. J. Lecomte, and D. A. Bryant, J. Nat. Prod. 71:1647-1650, 2008). Two genes that were predicted to encode enzymes involved in the biosynthesis of synechoxanthin were identified by comparative genomics, and these genes were insertionally inactivated in Synechococcus sp. strain PCC 7002 to verify their function. The cruE gene (SYNPCC7002_A1248) encodes β-carotene desaturase/methyltransferase, which converts β-carotene to renierapurpurin. The cruH gene (SYNPCC7002_A2246) encodes an enzyme that is minimally responsible for the hydroxylation/oxidation of the C-18 and C-18′ methyl groups of renierapurpurin. Based on observed and biochemically characterized intermediates, a complete pathway for synechoxanthin biosynthesis is proposed.     

Identification and characterization of a novel testis-specific gene CKT2, which encodes a substrate for protein kinase CK2

Protein kinase CK2 is a serine/threonine kinase known to phosphorylate numerous substrates. CK2 is implicated in several physiologic and pathologic processes, particularly in cancer biology. CK2 is comprised of several subunits, including CK2α, CK2α′ and CK2β. Inactivation of CK2α′ leads to chromatin degeneration of germ cells, resulting in male sterility. To identify additional targets of CK2α′ in testes and to determine the role of CK2α′ in germ cell nuclear integrity, GST pull-down and protein–protein interaction assays were conducted. A novel testis-specific gene, CKT2 (CK2 Target protein 2), was found whose product interacts with and is phosphorylated by CK2 in vitro and in vivo. CKT2 is a 30.2 kDa protein with one coiled-coil domain and six putative phosphorylation sites. High expression of CKT2 correlated with chromatin condensation of spermatids in murine testes. Findings reported herein demonstrate that CKT2 is a target protein of native CK2α′ in testes and suggest that CKT2 plays a role in chromatin regulation of male germ cells.     

AP endonucleases process 5-methylcytosine excision intermediates during active DNA demethylation in Arabidopsis

DNA methylation is a primary epigenetic modification regulating gene expression and chromatin structure in many eukaryotes. Plants have a unique DNA demethylation system in that 5-methylcytosine (5mC) is directly removed by DNA demethylases, such as DME/ROS1 family proteins, but little is known about the downstream events. During 5mC excision, DME produces 3′-phosphor-α, β-unsaturated aldehyde and 3′-phosphate by successive β- and δ-eliminations, respectively. The kinetic studies revealed that these 3′-blocking lesions persist for a significant amount of time and at least two different enzyme activities are required to immediately process them. We demonstrate that Arabidopsis AP endonucleases APE1L, APE2 and ARP have distinct functions to process such harmful lesions to allow nucleotide extension. DME expression is toxic to E. coli due to excessive 5mC excision, but expression of APE1L or ARP significantly reduces DME-induced cytotoxicity. Finally, we propose a model of base excision repair and DNA demethylation pathway unique to plants.     

Characterization of β-Ketoacyl-Acyl Carrier Protein Synthase III from Streptomyces glaucescens and Its Role in Initiation of Fatty Acid Biosynthesis

The Streptomyces glaucescens fabH gene, encoding β-ketoacyl-acyl carrier protein (β-ketoacyl-ACP) synthase (KAS) III (FabH), was overexpressed in Escherichia coli, and the resulting gene product was purified to homogeneity by metal chelate chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified protein revealed an M_r of 37,000, while gel filtration analysis determined a native M_r of 72,000 ± 3,000 (mean ± standard deviation), indicating that the enzyme is homodimeric. The purified recombinant protein demonstrated both KAS activity and acyl coenzyme A (acyl-CoA):ACP transacylase (ACAT) activity in a 1:0.12 ratio. The KAS and ACAT activities were both sensitive to thiolactomycin inhibition. The KAS activity of the protein demonstrated a K_m value of 3.66 μM for the malonyl-ACP substrate and an unusual broad specificity for acyl-CoA substrates, with K_m values of 2.4 μM for acetyl-CoA, 0.71 μM for butyryl-CoA, and 0.41 μM for isobutyryl-CoA. These data suggest that the S. glaucescens FabH is responsible for initiating both straight- and branched-chain fatty acid biosynthesis in Streptomyces and that the ratio of the various fatty acids produced by this organism will be dictated by the ratios of the various acyl-CoA substrates that can react with FabH. Results from a series of in vivo directed biosynthetic experiments in which the ratio of these acyl-CoA substrates was varied are consistent with this hypothesis. An additional set of in vivo experiments using thiolactomycin provides support for the role of FabH and further suggests that a FabH-independent pathway for straight-chain fatty acid biosynthesis operates in S. glaucescens.     

Novel Carotenoid Oxidase Involved in Biosynthesis of 4,4′-Diapolycopene Dialdehyde

Biosynthesis of C₃₀ carotenoids is relatively restricted in nature but has been described in Staphylococcus and in methylotrophic bacteria. We report here identification of a novel gene (crtNb) involved in conversion of 4,4′-diapolycopene to 4,4′-diapolycopene aldehyde. An aldehyde dehydrogenase gene (ald) responsible for the subsequent oxidation of 4,4′-diapolycopene aldehyde to 4,4′-diapolycopene acid was also identified in Methylomonas. CrtNb has significant sequence homology with diapophytoene desaturases (CrtN). However, data from knockout of crtNb and expression of crtNb in Escherichia coli indicated that CrtNb is not a desaturase but rather a novel carotenoid oxidase catalyzing oxidation of the terminal methyl group(s) of 4,4′-diaponeurosporene and 4,4′-diapolycopene to the corresponding terminal aldehyde. It has moderate to low activity on neurosporene and lycopene and no activity on β-carotene or ζ-carotene. Using a combination of C₃₀ carotenoid synthesis genes from Staphylococcus and Methylomonas, 4,4′-diapolycopene dialdehyde was produced in E. coli as the predominant carotenoid. This C₃₀ dialdehyde is a dark-reddish purple pigment that may have potential uses in foods and cosmetics.     

Batroxobin Binds Fibrin with Higher Affinity and Promotes Clot Expansion to a Greater Extent than Thrombin

Batroxobin is a thrombin-like serine protease from the venom of Bothrops atrox moojeni that clots fibrinogen. In contrast to thrombin, which releases fibrinopeptide A and B from the NH₂-terminal domains of the Aα- and Bβ-chains of fibrinogen, respectively, batroxobin only releases fibrinopeptide A. Because the mechanism responsible for these differences is unknown, we compared the interactions of batroxobin and thrombin with the predominant γ_A/γ_A isoform of fibrin(ogen) and the γ_A/γ′ variant with an extended γ-chain. Thrombin binds to the γ′-chain and forms a higher affinity interaction with γ_A/γ′-fibrin(ogen) than γ_A/γ_A-fibrin(ogen). In contrast, batroxobin binds both fibrin(ogen) isoforms with similar high affinity (K_d values of about 0.5 μm) even though it does not interact with the γ′-chain. The batroxobin-binding sites on fibrin(ogen) only partially overlap with those of thrombin because thrombin attenuates, but does not abrogate, the interaction of γ_A/γ_A-fibrinogen with batroxobin. Furthermore, although both thrombin and batroxobin bind to the central E-region of fibrinogen with a K_d value of 2–5 μm, the α(17–51) and Bβ(1–42) regions bind thrombin but not batroxobin. Once bound to fibrin, the capacity of batroxobin to promote fibrin accretion is 18-fold greater than that of thrombin, a finding that may explain the microvascular thrombosis that complicates envenomation by B. atrox moojeni. Therefore, batroxobin binds fibrin(ogen) in a manner distinct from thrombin, which may contribute to its higher affinity interaction, selective fibrinopeptide A release, and prothrombotic properties.     

Synthesis,properties, and biological activity of boranophosphate analogs of the mRNA cap: versatile tools for manipulation of therapeutically relevant cap-dependent processes

Modified mRNA cap analogs aid in the study of mRNA-related processes and may enable creation of novel therapeutic interventions. We report the synthesis and properties of 11 dinucleotide cap analogs bearing a single boranophosphate modification at either the α-, β- or γ-position of the 5′,5′-triphosphate chain. The compounds can potentially serve either as inhibitors of translation in cancer cells or reagents for increasing expression of therapeutic proteins in vivo from exogenous mRNAs. The BH₃-analogs were tested as substrates and binding partners for two major cytoplasmic cap-binding proteins, DcpS, a decapping pyrophosphatase, and eIF4E, a translation initiation factor. The susceptibility to DcpS was different between BH₃-analogs and the corresponding analogs containing S instead of BH₃ (S-analogs). Depending on its placement, the boranophosphate group weakened the interaction with DcpS but stabilized the interaction with eIF4E. The first of the properties makes the BH₃-analogs more stable and the second, more potent as inhibitors of protein biosynthesis. Protein expression in dendritic cells was 2.2- and 1.7-fold higher for mRNAs capped with m₂^7,2′-OGpp_BH3pG D1 and m₂^7,2′-OGpp_BH3pG D2, respectively, than for in vitro transcribed mRNA capped with m₂^7,3′-OGpppG. Higher expression of cancer antigens would make mRNAs containing m₂^7,2′-OGpp_BH3pG D1 and m₂^7,2′-OGpp_BH3pG D2 favorable for anticancer immunization.     

Proofreading exonuclease on a tether: the complex between the E. coli DNA polymerase III subunits α, ε, θ and β reveals a highly flexible arrangement of the proofreading domain

A complex of the three (αεθ) core subunits and the β₂ sliding clamp is responsible for DNA synthesis by Pol III, the Escherichia coli chromosomal DNA replicase. The 1.7 Å crystal structure of a complex between the PHP domain of α (polymerase) and the C-terminal segment of ε (proofreading exonuclease) subunits shows that ε is attached to α at a site far from the polymerase active site. Both α and ε contain clamp-binding motifs (CBMs) that interact simultaneously with β₂ in the polymerization mode of DNA replication by Pol III. Strengthening of both CBMs enables isolation of stable αεθ:β₂ complexes. Nuclear magnetic resonance experiments with reconstituted αεθ:β₂ demonstrate retention of high mobility of a segment of 22 residues in the linker that connects the exonuclease domain of ε with its α-binding segment. In spite of this, small-angle X-ray scattering data show that the isolated complex with strengthened CBMs has a compact, but still flexible, structure. Photo-crosslinking with p-benzoyl-L-phenylalanine incorporated at different sites in the α-PHP domain confirm the conformational variability of the tether. Structural models of the αεθ:β₂ replicase complex with primer-template DNA combine all available structural data.     

Substrate Specificity of Purified Recombinant Human β-Carotene 15,15′-Oxygenase (BCO1)

Humans cannot synthesize vitamin A and thus must obtain it from their diet. β-Carotene 15,15′-oxygenase (BCO1) catalyzes the oxidative cleavage of provitamin A carotenoids at the central 15–15′ double bond to yield retinal (vitamin A). In this work, we quantitatively describe the substrate specificity of purified recombinant human BCO1 in terms of catalytic efficiency values (k_cat/K_m). The full-length open reading frame of human BCO1 was cloned into the pET-28b expression vector with a C-terminal polyhistidine tag, and the protein was expressed in the Escherichia coli strain BL21-Gold(DE3). The enzyme was purified using cobalt ion affinity chromatography. The purified enzyme preparation catalyzed the oxidative cleavage of β-carotene with a V_max = 197.2 nmol retinal/mg BCO1 × h, K_m = 17.2 μm and catalytic efficiency k_cat/K_m = 6098 m⁻¹ min⁻¹. The enzyme also catalyzed the oxidative cleavage of α-carotene, β-cryptoxanthin, and β-apo-8′-carotenal to yield retinal. The catalytic efficiency values of these substrates are lower than that of β-carotene. Surprisingly, BCO1 catalyzed the oxidative cleavage of lycopene to yield acycloretinal with a catalytic efficiency similar to that of β-carotene. The shorter β-apocarotenals (β-apo-10′-carotenal, β-apo-12′-carotenal, β-apo-14′-carotenal) do not show Michaelis-Menten behavior under the conditions tested. We did not detect any activity with lutein, zeaxanthin, and 9-cis-β-carotene. Our results show that BCO1 favors full-length provitamin A carotenoids as substrates, with the notable exception of lycopene. Lycopene has previously been reported to be unreactive with BCO1, and our findings warrant a fresh look at acycloretinal and its alcohol and acid forms as metabolites of lycopene in future studies.