Eiko Kanaya

Activation of Orphan Nuclear Constitutive Androstane Receptor Requires Subnuclear Targeting by Peroxisome Proliferator-activated Receptor γ Coactivator-1α A POSSIBLE LINK BETWEEN XENOBIOTIC RESPONSE AND NUTRITIONAL STATE

In contrast to the classical nuclear receptors, the constitutive androstane receptor (CAR) is transcriptionally active in the absence of ligand. In the course of searching for the mediator of CAR activation, we found that ligand-independent activation of CAR was achieved in cooperation with the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). PGC-1β, a PGC-1α homologue, also activated CAR to less of an extent than PGC-1α. Coexpression of the ligand-binding domain of a heterodimerization partner, retinoid X receptor α, enhanced the PGC-1α-mediated activation of CAR, although it had a weak effect on the basal activity of CAR in the absence of PGC-1α. Both the N-terminal region, with the LXXLL motif, and the C-terminal region, with a serine/arginine-rich domain (RS domain), in PGC-1α were required for full activation of CAR. Pull-down experiments using recombinant proteins revealed that CAR directly interacted with both the LXXLL motif and the RS domain. Furthermore, we demonstrated that the RS domain of PGC-1α was required for CAR localization at nuclear speckles. These results indicate that PGC-1α mediates the ligand-independent activation of CAR by means of subnuclear targeting through the RS domain of PGC-1α.

The nuclear bile acid receptor FXR is activated by PGC-1α in a ligand-dependent manner

The nuclear bile acid receptor FXR (farnesoid X receptor) is one of the key factors that suppress bile acid biosynthesis in the liver. PGC-1alpha [PPARgamma (peroxisome-proliferator-activated receptor gamma) co-activator-1alpha] is known to control energy homoeostasis in adipose tissue, skeletal muscle and liver. We performed cell-based reporter assays using the expression system of a GAL4-FXR chimaera, the ligand-binding domain of FXR fused to the DNA-binding domain of yeast GAL4, to find the co-activators for FXR. We found that the transcriptional activation of a reporter plasmid by a GAL4-FXR chimaera was strongly enhanced by PGC-1alpha, in a ligand-dependent manner. Transcriptional activation of the SHP (small heterodimer partner) gene by the FXR-RXRalpha (retinoid X receptor alpha) heterodimer was also enhanced by PGC-1alpha in the presence of CDCA (chenodeoxycholic acid). Co-immunoprecipitation and pull-down studies using glutathione S-transferase-PGC-1alpha fusion proteins revealed that the ligand-binding domain of FXR binds PGC-1alpha in a ligand-influenced manner both in vivo and in vitro. Furthermore, our studies revealed that SHP represses its own transcription, and the addition of excess amounts of PGC-1alpha can overcome the inhibitory effect of SHP. These observations indicate that PGC-1alpha mediates the ligand-dependent activation of FXR and transcription of SHP gene.

Isolation of a Novel Cutinase Homolog with Polyethylene Terephthalate-Degrading Activity from Leaf-Branch Compost by Using a Metagenomic Approach

ABSTRACT The gene encoding a cutinase homolog, LC-cutinase, was cloned from a fosmid library of a leaf-branch compost metagenome by functional screening using tributyrin agar plates. LC-cutinase shows the highest amino acid sequence identity of 59.7% to Thermomonospora curvata lipase. It also shows the 57.4% identity to Thermobifida fusca cutinase. When LC-cutinase without a putative signal peptide was secreted to the periplasm of Escherichia coli cells with the assistance of the pelB leader sequence, more than 50% of the recombinant protein, termed LC-cutinase*, was excreted into the extracellular medium. It was purified and characterized. LC-cutinase* hydrolyzed various fatty acid monoesters with acyl chain lengths of 2 to 18, with a preference for short-chain substrates (C4 substrate at most) most optimally at pH 8.5 and 50°C, but could not hydrolyze olive oil. It lost activity with half-lives of 40 min at 70°C and 7 min at 80°C. LC-cutinase* had an ability to degrade poly(ε-caprolactone) and polyethylene terephthalate (PET). The specific PET-degrading activity of LC-cutinase* was determined to be 12 mg/h/mg of enzyme (2.7 mg/h/μkat of pNP-butyrate-degrading activity) at pH 8.0 and 50°C. This activity is higher than those of the bacterial and fungal cutinases reported thus far, suggesting that LC-cutinase* not only serves as a good model for understanding the molecular mechanism of PET-degrading enzyme but also is potentially applicable for surface modification and degradation of PET.

Crystal structure and thermodynamic and kinetic stability of metagenome-derived LC-cutinase.

The crystal structure of metagenome-derived LC-cutinase with polyethylene terephthalate (PET)-degrading activity was determined at 1.5 Å resolution. The structure strongly resembles that of Thermobifida alba cutinase. Ser165, Asp210, and His242 form the catalytic triad. Thermal denaturation and guanidine hydrochloride (GdnHCl)-induced unfolding of LC-cutinase were analyzed at pH 8.0 by circular dichroism spectroscopy. The midpoint of the transition of the thermal denaturation curve, T1/2, and that of the GdnHCl-induced unfolding curve, Cm, at 30 °C were 86.2 °C and 4.02 M, respectively. The free energy change of unfolding in the absence of GdnHCl, ΔG(H2O), was 41.8 kJ mol(-1) at 30 °C. LC-cutinase unfolded very slowly in GdnHCl with an unfolding rate, ku(H2O), of 3.28 × 10(-6) s(-1) at 50 °C. These results indicate that LC-cutinase is a kinetically robust protein. Nevertheless, the optimal temperature for the activity of LC-cutinase toward p-nitrophenyl butyrate (50 °C) was considerably lower than the T1/2 value. It increased by 10 °C in the presence of 1% polyethylene glycol (PEG) 1000. It also increased by at least 20 °C when PET was used as a substrate. These results suggest that the active site is protected from a heat-induced local conformational change by binding of PEG or PET. LC-cutinase contains one disulfide bond between Cys275 and Cys292. To examine whether this disulfide bond contributes to the thermodynamic and kinetic stability of LC-cutinase, C275/292A-cutinase without this disulfide bond was constructed. Thermal denaturation studies and equilibrium and kinetic studies of the GdnHCl-induced unfolding of C275/292A-cutinase indicate that this disulfide bond contributes not only to the thermodynamic stability but also to the kinetic stability of LC-cutinase.

Flavobacterium banpakuense sp. nov., isolated from leaf-and-branch compost.

A strictly aerobic, Gram-stain-negative, yellow-pigmented, non-spore-forming, motile (by gliding), rod-shaped bacterium, designated strain 15F3(T), was isolated from leaf-and-branch compost. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 15F3(T) was most closely related to Flavobacterium reichenbachii WB 3.2-61(T) and formed a distinct phyletic lineage within the genus Flavobacterium, the type genus of the family Flavobacteriaceae. Growth was observed at 10-34 °C (optimum, 30 °C) and pH 6.0-8.0 (optimum, pH 7.0). No growth occurred in the presence of ≥2 % (w/v) NaCl. Strain 15F3(T) reduced nitrate to nitrogen and showed catalase activity but no oxidase activity. The predominant cellular fatty acids were iso-C(15 : 0) and summed feature 3 (comprising C(16 : 1)ω7c and/or iso-C(15 : 0) 2-OH). The major isoprenoid quinone was menaquinone-6. The G+C content of the genomic DNA was 31.1 mol%. On the basis of data from this polyphasic study, strain 15F3(T) may be classified as a representative of a novel species within the genus Flavobacterium, for which the name Flavobacterium banpakuense sp. nov. is proposed; the type strain is 15F3(T) ( = KACC 14225(T)  = JCM 16466(T)).

Flavobacterium compostarboris sp. nov., isolated from leaf-and-branch compost, and emended descriptions of Flavobacterium hercynium, Flavobacterium resistens and Flavobacterium johnsoniae

A strictly aerobic, Gram-negative, yellow-pigmented, non-spore-forming rod, designated 15C3(T), was isolated from aerobic leaf-and-branch compost at EXPO Park in Osaka, Japan. Growth was observed at 9-33 °C (optimum 25 °C) and pH 5.6-7.9 (optimum pH 6.1-7.0). No growth occurred with >2% (w/v) NaCl. Strain 15C3(T) reduced nitrate to nitrogen and showed catalase activity but not oxidase activity. The predominant fatty acids were iso-C(15:0), iso-C(17:0) 3-OH and summed feature 3 (comprising C(16:1)ω7c and/or iso-C(15:0) 2-OH). The isolate contained phosphatidylethanolamine as the major polar lipid and menaquinone-6 as the major respiratory quinone. The G+C content of the genomic DNA of strain 15C3(T) was 33.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 15C3(T) belonged to the genus Flavobacterium and was most closely related to Flavobacterium hercynium WB 4.2-33(T) (96.9% sequence similarity). On the basis of phenotypic and phylogenetic distinctiveness, strain 15C3(T) is considered to represent a novel species in the genus Flavobacterium, for which the name Flavobacterium compostarboris sp. nov. is proposed. The type strain is 15C3(T) ( = KACC 14224(T)  = JCM 16527(T)). Emended descriptions of F. hercynium, Flavobacterium resistens and Flavobacterium johnsoniae are also given.

Structure and stability of metagenome-derived glycoside hydrolase family 12 cellulase (LC-CelA) a homolog of Cel12A from Rhodothermus marinus☆

Ten genes encoding novel cellulases with putative signal peptides at the N‐terminus, termed pre‐LC‐CelA–J, were isolated from a fosmid library of a leaf–branch compost metagenome by functional screening using agar plates containing carboxymethyl cellulose and trypan blue. All the cellulases except pre‐LC‐CelG have a 14–29 residue long flexible linker (FL) between the signal peptide and the catalytic domain. LC‐CelA without a signal peptide (residues 20–261), which shows 76% amino acid sequence identity to Cel12A from Rhodothermus marinus (RmCel12A), was overproduced in Escherichia coli, purified and characterized. LC‐CelA exhibited its highest activity across a broad pH range (pH 5–9) and at 90 °C, indicating that LC‐CelA is a highly thermostable cellulase, like RmCel12A. The crystal structure of LC‐CelA was determined at 1.85 Å resolution and is nearly identical to that of RmCel12A determined in a form without the FL. Both proteins contain two disulfide bonds. LC‐CelA has a 16‐residue FL (residues 20–35), most of which is not visible in the electron density map, probably due to structural disorder. However, Glu34 and Pro35 form hydrogen bonds with the central region of the protein. ΔFL‐LC‐CelA (residues 36–261) and E34A‐LC‐CelA with a single Glu34 → Ala mutation were therefore constructed and characterized. ΔFL‐LC‐CelA and E34A‐LC‐CelA had lower melting temperatures (T m) than LC‐CelA by 14.7 and 12.0 °C respectively. The T m of LC‐CelA was also decreased by 28.0 °C in the presence of dithiothreitol. These results suggest that Glu34‐mediated hydrogen bonds and the two disulfide bonds contribute to the stabilization of LC‐CelA.

The N-terminal hybrid binding domain of RNase HI from Thermotoga maritima is important for substrate binding and Mg2+-dependent activity

Thermotoga maritima ribonuclease H (RNase H) I (Tma‐RNase HI) contains a hybrid binding domain (HBD) at the N‐terminal region. To analyze the role of this HBD, Tma‐RNase HI, Tma‐W22A with the single mutation at the HBD, the C‐terminal RNase H domain (Tma‐CD) and the N‐terminal domain containing the HBD (Tma‐ND) were overproduced in Escherichia coli, purified and biochemically characterized. Tma‐RNase HI prefers Mg2+ to Mn2+ for activity, and specifically loses most of the Mg2+‐dependent activity on removal of the HBD and 87% of it by the mutation at the HBD. Tma‐CD lost the ability to suppress the RNase H deficiency of an E. coli rnhA mutant, indicating that the HBD is responsible for in vivo RNase H activity. The cleavage‐site specificities of Tma‐RNase HI are not significantly changed on removal of the HBD, regardless of the metal cofactor. Binding analyses of the proteins to the substrate using surface plasmon resonance indicate that the binding affinity of Tma‐RNase HI is greatly reduced on removal of the HBD or the mutation. These results indicate that there is a correlation between Mg2+‐dependent activity and substrate binding affinity. Tma‐CD was as stable as Tma‐RNase HI, indicating that the HBD is not important for stability. The HBD of Tma‐RNase HI is important not only for substrate binding, but also for Mg2+‐dependent activity, probably because the HBD affects the interaction between the substrate and enzyme at the active site, such that the scissile phosphate group of the substrate and the Mg2+ ion are arranged ideally.

Role of RNase H1 in DNA repair: removal of single ribonucleotide misincorporated into DNA in collaboration with RNase H2

Several RNases H1 cleave the RNA-DNA junction of Okazaki fragment-like RNA-DNA/DNA substrate. This activity, termed 3’-junction ribonuclease (3’-JRNase) activity, is different from the 5’-JRNase activity of RNase H2 that cleaves the 5’-side of the ribonucleotide of the RNA-DNA junction and is required to initiate the ribonucleotide excision repair pathway. To examine whether RNase H1 exhibits 3’-JRNase activity for dsDNA containing a single ribonucleotide and can remove this ribonucleotide in collaboration with RNase H2, cleavage of a DNA8-RNA1-DNA9/DNA18 substrate with E. coli RNase H1 and H2 was analyzed. This substrate was cleaved by E. coli RNase H1 at the (5’)RNA-DNA(3’) junction, regardless of whether it was cleaved by E. coli RNase H2 at the (5’)DNA-RNA(3’) junction in advance or not. Likewise, this substrate was cleaved by E. coli RNase H2 at the (5’)DNA-RNA(3’) junction, regardless of whether it was cleaved by E. coli RNase H1 at the (5’)RNA-DNA(3’) junction in advance or not. When this substrate was cleaved by a mixture of E. coli RNases H1 and H2, the ribonucleotide was removed from the substrate. We propose that RNase H1 is involved in the excision of single ribonucleotides misincorporated into DNA in collaboration with RNase H2.

Activity, stability, and structure of metagenome-derived LC11-RNase H1, a homolog of Sulfolobus tokodaii RNase H1.

Metagenome‐derived LC11‐RNase H1 is a homolog of Sulfolobus tokodaii RNase H1 (Sto‐RNase H1). It lacks a C‐terminal tail, which is responsible for hyperstabilization of Sto‐RNase H1. Sto‐RNase H1 is characterized by its ability to cleave not only an RNA/DNA hybrid but also a double‐stranded RNA (dsRNA). To examine whether LC11‐RNase H1 also exhibits both RNase H and dsRNase activities, LC11‐RNase H1 was overproduced in Escherichia coli, purified, and characterized. LC11‐RNase H1 exhibited RNase H activity with similar metal ion preference, optimum pH, and cleavage mode of substrate with those of Sto‐RNase H1. However, LC11‐RNase H1 did not exhibit dsRNase activity at any condition examined. LC11‐RNase H1 was less stable than Sto‐RNases H1 and its derivative lacking the C‐terminal tail (Sto‐RNase H1ΔC6) by 37 and 13°C in Tm, respectively. To understand the structural bases for these differences, the crystal structure of LC11‐RNase H1 was determined at 1.4 Å resolution. The LC11‐RNase H1 structure is highly similar to the Sto‐RNase H1 structure. However, LC11‐RNase H1 has two grooves on protein surface, one containing the active site and the other containing DNA‐phosphate binding pocket, while Sto‐RNase H1 has one groove containing the active site. In addition, LC11‐RNase H1 contains more cavities and buried charged residues than Sto‐RNase H1. We propose that LC11‐RNase H1 does not exhibit dsRNase activity because dsRNA cannot fit to the two grooves on protein surface and that LC11‐RNase H1 is less stable than Sto‐RNase H1ΔC6 because of the increase in cavity volume and number of buried charged residues.