Kon Ho Lee

RshA, an anti-sigma factor that regulates the activity of the mycobacterial stress response sigma factor SigH

SigH, an alternative sigma factor of Mycobacterium tuberculosis,  is  a  central regulator  of  the  response to oxidative and heat stress. Exposure to these stresses results in increased expression of sigH itself, and of genes encoding additional regulators and effectors of the bacterial response to these stresses. In this work we show that RshA, a protein encoded by a gene in the sigH operon, is an anti‐sigma factor of SigH. We demonstrate that RshA binds to SigH in vitro and in vivo. This protein–protein interaction, as  well  as the  ability  of RshA to inhibit SigH‐dependent transcription, is redox‐dependent, with RshA functioning as a negative regulator of SigH activity only under reducing conditions. The interaction of SigH and RshA is also disrupted in vitro by elevated temperature. RshA, a protein of 101 amino acids, contains five conserved cysteine residues of which two appear to be essential for RshA to inhibit SigH activity, suggesting that these cysteines may be important for the redox state dependence of RshA function. Our results indicate that RshA is a sensor that responds to oxidative stress, and also to heat stress, resulting in activation of SigH and expression of the SigH‐dependent genes that allow M. tuberculosis to adapt to these stresses.

Geranylated flavonoids displaying SARS-CoV papain-like protease inhibition from the fruits of Paulownia tomentosa

Abstract SARS-CoV papain-like protease (PLpro) is an important antiviral target due to its key roles in SARS virus replication. The MeOH extracts of the fruits of the Paulownia tree yielded many small molecules capable of targeting PLpro. Five of these compounds were new geranylated flavonoids, tomentin A, tomentin B, tomentin C, tomentin D, tomentin E (1–5). Structure analysis of new compounds (1–5) by NMR showed that they all contain a 3,4-dihydro-2H-pyran moiety. This chemotype is very rare and is derived from cyclization of a geranyl group with a phenol functionality. Most compounds (1–12) inhibited PLpro in a dose dependent manner with IC50’s raging between 5.0 and 14.4μM. All new compounds having the dihydro-2H-pyran group showed better inhibition than their parent compounds (1 vs 11, 2 vs 9, 4 vs 12, 5 vs 6). In kinetic studies, 1–12 emerged to be reversible, mixed inhibitors.

A Ds-insertion mutant of OSH6 (Oryza sativa Homeobox 6) exhibits outgrowth of vestigial leaf-like structures, bracts, in rice

OSH6 (Oryza sativa Homeobox6) is an ortholog of lg3 (Liguleless3) in maize. We generated a novel allele, termed OSH6-Ds, by inserting a defective Ds element into the third exon of OSH6, which resulted in a truncated OSH6 mRNA. The truncated mRNA was expressed ectopically in leaf tissues and encoded the N-terminal region of OSH6, which includes the KNOX1 and partial KNOX2 subdomains. This recessive mutant showed outgrowth of bracts or produced leaves at the basal node of the panicle. These phenotypes distinguished it from the OSH6 transgene whose ectopic expression led to a “blade to sheath transformation” phenotype at the midrib region of leaves, similar to that seen in dominant Lg3 mutants. Expression of a similar truncated OSH6 cDNA from the 35S promoter (35S::ΔOSH6) confirmed that the ectopic expression of this product was responsible for the aberrant bract development. These data suggest that OSH6-Ds interferes with a developmental mechanism involved in bract differentiation, especially at the basal nodes of panicles.

Engineering of Family-5 Glycoside Hydrolase (Cel5A) from an Uncultured Bacterium for Efficient Hydrolysis of Cellulosic Substrates

Cel5A, an endoglucanase, was derived from the metagenomic library of vermicompost. The deduced amino acid sequence of Cel5A shows high sequence homology with family-5 glycoside hydrolases, which contain a single catalytic domain but no distinct cellulose-binding domain. Random mutagenesis and cellulose-binding module (CBM) fusion approaches were successfully applied to obtain properties required for cellulose hydrolysis. After two rounds of error-prone PCR and screening of 3,000 mutants, amino acid substitutions were identified at various positions in thermotolerant mutants. The most heat-tolerant mutant, Cel5A_2R2, showed a 7-fold increase in thermostability. To enhance the affinity and hydrolytic activity of Cel5A on cellulose substrates, the family-6 CBM from Saccharophagus degradans was fused to the C-terminus of the Cel5A_2R2 mutant using overlap PCR. The Cel5A_2R2-CBM6 fusion protein showed 7-fold higher activity than the native Cel5A on Avicel and filter paper. Cellobiose was a major product obtained from the hydrolysis of cellulosic substrates by the fusion enzyme, which was identified by using thin layer chromatography analysis.

Functional characterization of orchardgrass endoplasmic reticulum-resident Hsp90 (DgHsp90) as a chaperone and an ATPase.

Hsp90 proteins are essential molecular chaperones regulating multiple cellular processes in distinct subcellular organelles. In this study, we report the functional characterization of a cDNA encoding endoplasmic reticulum (ER)-resident Hsp90 from orchardgrass (DgHsp90). DgHsp90 is a 2742bp cDNA with an open reading frame predicted to encode an 808 amino acid protein. DgHsp90 has a well conserved N-terminal ATPase domain and a C-terminal Hsp90 domain and ER-retention motif. Expression of DgHsp90 increased during heat stress at 35 degrees C or H(2)O(2) treatment. DgHsp90 also functions as a chaperone protein by preventing thermal aggregation of malate dehydrogenase (EC 1.1.1.37) and citrate synthase (EC 2.3.3.1). The intrinsic ATPase activity of DgHsp90 was inhibited by geldanamycin, an Hsp90 inhibitor, and the inhibition reduced the chaperone activity of DgHsp90. Yeast cells overexpressing DgHsp90 exhibited enhanced thermotolerance.

Structure of Chlorobium tepidum Sepiapterin Reductase Complex Reveals the Novel Substrate Binding Mode for Stereospecific Production of l-threo-Tetrahydrobiopterin

Sepiapterin reductase (SR) is involved in the last step of tetrahydrobiopterin (BH4) biosynthesis by reducing the di-keto group of 6-pyruvoyl tetrahydropterin. Chlorobium tepidum SR (cSR) generates a distinct BH4 product, l-threo-BH4 (6R-(1′S,2′S)-5,6,7,8-BH4), whereas animal enzymes produce l-erythro-BH4 (6R-(1′R,2′S)-5,6,7,8-BH4) although it has high amino acid sequence similarities to the other animal enzymes. To elucidate the structural basis for the different reaction stereospecificities, we have determined the three-dimensional structures of cSR alone and complexed with NADP and sepiapterin at 2.1 and 1.7 Å resolution, respectively. The overall folding of the cSR, the binding site for the cofactor NADP(H), and the positions of active site residues were quite similar to the mouse and the human SR. However, significant differences were found in the substrate binding region of the cSR. In comparison to the mouse SR complex, the sepiapterin in the cSR is rotated about 180° around the active site and bound between two aromatic side chains of Trp-196 and Phe-99 so that its pterin ring is shifted to the opposite side, but its side chain position is not changed. The swiveled sepiapterin binding results in the conversion of the side chain configuration, exposing the opposite face for hydride transfer from NADPH. The different sepiapterin binding mode within the conserved catalytic architecture presents a novel strategy of switching the reaction stereospecificities in the same protein fold.

Identification and characterization of the second cysteine protease inhibitor of Clonorchis sinensis (CsStefin-2)

CsStefin-2, the second cysteine protease inhibitor of Clonorchis sinensis, was identified and characterized. CsStefin-2 is a cysteine protease inhibitor that belongs to family 1 stefins based on its phylogenetic and structural properties. However, CsStefin-2 had a QIVSG cystatin motif distinct from the common QVVAG cystatin motif that is well conserved in family 1 stefins. Mutagenesis analysis revealed that the two amino acid substitutions in the QIVSG cystatin motif of CsStefin-2 did not affect its inhibitory activity. Molecular modeling also indicated that no critical change was induced in the interaction between CsStefin-2 and its target enzyme. CsStefin-2 showed broad inhibitory activities against several cysteine proteases, including human cathepsins B and L, papain, and cathepsin Fs of C. sinensis (CsCFs), and effectively inhibited the autocatalytic maturation of CsCF-6. Native CsStefin-2 was assembled into a homo-tetramer, in which intermolecular disulfide bonds are not involved in the assembly of the tetramer. CsStefin-2 was expressed throughout the various developmental stages of the parasite and was localized in the intestinal epithelium, where CsCFs are actively synthesized. These results suggest that CsStefin-2 is the second active cysteine protease inhibitor of C. sinensis that shares functional redundancy with CsStefin-1 to modulate the activity and processing of CsCFs.

Phenolic phytochemical displaying SARS-CoV papain-like protease inhibition from the seeds of Psoralea corylifolia

Abstract Severe acute respiratory syndrome coronavirus (SARS-CoV) papain-like protease (PLpro) is a key enzyme that plays an important role in SARS virus replication. The ethanol extract of the seeds of Psoralea corylifolia showed high activity against the SARS-CoV PLpro with an IC50 of value of 15 µg/ml. Due to its potency, subsequent bioactivity-guided fractionation of the ethanol extract led to six aromatic compounds (1–6), which were identified as bavachinin (1), neobavaisoflavone (2), isobavachalcone (3), 4′-O-methylbavachalcone (4), psoralidin (5) and corylifol A (6). All isolated flavonoids (1–6) inhibited PLpro in a dose-dependent manner with IC50 ranging between 4.2 and 38.4 µM. Lineweaver–Burk and Dixon plots and their secondary replots indicated that inhibitors (1–6) were mixed inhibitors of PLpro. The analysis of KI and KIS values proved that the two most promising compounds (3 and 5) had reversible mixed type I mechanisms.

6-Pyruvoyltetrahydropterin synthase orthologs of either a single or dual domain structure are responsible for tetrahydrobiopterin synthesis in bacteria

6‐Pyruvoyltetrahydropterin synthase (PTPS) catalyzes the second step of tetrahydrobiopterin (BH4) synthesis. We previously identified PTPS orthologs (bPTPS‐Is) in bacteria which do not produce BH4. In this study we disrupted the gene encoding bPTPS‐I in Synechococcus sp. PCC 7942, which produces BH4‐glucoside. The mutant was normal in BH4‐glucoside production, demonstrating that bPTPS‐I does not participate in BH4 synthesis in vivo and bringing us a new PTPS ortholog (bPTPS‐II) of a bimodular polypeptide. The recombinant Synechococcus bPTPS‐II was assayed in vitro to show PTPS activity higher than human enzyme. Further computational analysis revealed the presence of mono and bimodular bPTPS‐II orthologs mostly in green sulfur bacteria and cyanobacteria, respectively, which are well known for BH4‐glycoside production. In summary we found new bacterial PTPS orthologs, having either a single or dual domain structure and being responsible for BH4 synthesis in vivo, thereby disclosing all the bacterial PTPS homologs.

A new tip homolog, ShTIP, from Salicornia shows a different involvement in salt stress compared to that of TIP from Arabidopsis

To obtain an insight into the comprehensive molecular characteristics of the salt tolerance mechanism, we performed a screening for salt inducible genes in a halophytic plant, Salicornia herbacea, using mRNA differential display. A comparative analysis of gene expression in Salicornia grown in control and salt-stressed conditions led to the detection of a gene that was induced by salt. Both sequence analysis and a subsequent database search revealed that this gene was highly homologous to tonoplast intrinsic proteins (TIPs) from a variety of plant species. This gene, designated as ShTIP, is 1014 bp in size and contains a coding region of 762 nucleotides, which encodes a protein of 254 amino acids. Northern blot analysis revealed that ShTIP was predominantly expressed in shoots under normal conditions. However, salt stress induced high expression of ShTIP in both the shoots and roots. The expression of ShTIP in a salt-sensitive calcineurin-deficient yeast mutant (cnbΔ) resulted in a resistance to the high salt conditions. In addition, we compared the expression of a TIP gene in Arabidopsis with that of ShTIP under different conditions and found that the Salicornia TIP has a different regulatory mechanism for adapting to salt stress conditions compared with the glycophyte Arabidopsis TIP. These results indicate that ShTIP plays an important role in salt tolerance.