Nazalan Najimudin

Draft genome sequence of the rubber tree Hevea brasiliensis

BackgroundHevea brasiliensis, a member of the Euphorbiaceae family, is the major commercial source of natural rubber (NR). NR is a latex polymer with high elasticity, flexibility, and resilience that has played a critical role in the world economy since 1876.ResultsHere, we report the draft genome sequence of H. brasiliensis. The assembly spans ~1.1 Gb of the estimated 2.15 Gb haploid genome. Overall, ~78% of the genome was identified as repetitive DNA. Gene prediction shows 68,955 gene models, of which 12.7% are unique to Hevea. Most of the key genes associated with rubber biosynthesis, rubberwood formation, disease resistance, and allergenicity have been identified.ConclusionsThe knowledge gained from this genome sequence will aid in the future development of high-yielding clones to keep up with the ever increasing need for natural rubber.

Vipirinin, a Coumarin-based HIV-1 Vpr Inhibitor, Interacts with a Hydrophobic Region of VPR

The human immunodeficiency virus 1 (HIV-1) viral protein R (Vpr) is an accessory protein that has been shown to have multiple roles in HIV-1 pathogenesis. By screening chemical libraries in the RIKEN Natural Products Depository, we identified a 3-phenyl coumarin-based compound that inhibited the cell cycle arrest activity of Vpr in yeast and Vpr-dependent viral infection of human macrophages. We determined its minimal pharmacophore through a structure-activity relationship study and produced more potent derivatives. We detected direct binding, and by assaying a panel of Vpr mutants, we found the hydrophobic region about residues Glu-25 and Gln-65 to be potentially involved in the binding of the inhibitor. Our findings exposed a targeting site on Vpr and delineated a convenient approach to explore other targeting sites on the protein using small molecule inhibitors as bioprobes.

Phylogeny and Characterization of Three nifH-Homologous Genes from Paenibacillus azotofixans

ABSTRACT In this paper, we report the cloning and characterization of three Paenibacillus azotofixans DNA regions containing genes involved in nitrogen fixation. Sequencing analysis revealed the presence of nifB1H1D1K1 gene organization in the 4,607-bp SacI DNA fragment. This is the first report of linkage of a nifB open reading frame upstream of the structural nif genes. The second (nifB2H2) and third (nifH3) nif homologues are confined within the 6,350-bp HindIII and 2,840-bp EcoRI DNA fragments, respectively. Phylogenetic analysis demonstrated that NifH1 and NifH2 form a monophyletic group among cyanobacterial NifH proteins. NifH3, on the other hand, clusters among NifH proteins of the highly divergent methanogenic archaea.

Production of poly(3-hydroxybutyrate) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Erwinia sp. USMI-20.

A locally isolated soil microorganism identified as Erwinia sp. USMI-20 has been found to produce poly(3-hydroxybutyrate), P(3HB), from either palm oil or glucose and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(3HB-co-3HV), from a combination of palm oil and a second carbon source of either one of the following compounds: propionic acid, n-propanol, valeric acid and n-pentanol. It was found that Erwinia sp. USMI-20 could produce P(3HB) up to 69 wt.% polymer content with a dry cell weight of 4.4 g/l from an initial amount of 14.5 g/l of glucose followed by a feeding rate of glucose at 0.48 g/h glucose. On the other hand, the bacteria can achieve 46 wt.% of P(3HB) and a dry cell weight of 3.6 g/l from a batch fermentation in a 10-l fermentor from an initial concentration of 4.6 g/l of palm oil. Further characterisation of the polymer production was also carried out by using different types of palm oil. Among the different palm oils that were used, crude palm oil was the best lipid source for P(3HB) production as compared to palm olein and palm kernel oil. In the production of the copolymer, P(3HB-co-3HV), the highest mole fraction of 3-HV units could be as high as 47 mol% from a single feeding of valeric acid upon initial growth on palm oil.

Characterization of Afp1, an antifreeze protein from the psychrophilic yeast Glaciozyma antarctica PI12.

The psychrophilic yeast Glaciozyma antarctica demonstrated high antifreeze activity in its culture filtrate. The culture filtrate exhibited both thermal hysteresis (TH) and ice recrystallization inhibition (RI) properties. The TH of 0.1xa0°C was comparable to that previously reported for bacteria and fungi. A genome sequence survey of the G. antarctica genome identified a novel antifreeze protein gene. The cDNA encoded a 177 amino acid protein with 30xa0% similarity to a fungal antifreeze protein from Typhula ishikariensis. The expression levels of AFP1 were quantified via real time-quantitative polymerase chain reaction (RT-qPCR), and the highest expression levels were detected within 6xa0h of growth at −12xa0°C. The cDNA of the antifreeze protein was cloned into an Escherichia coli expression system. Expression of recombinant Afp1 in E. coli resulted in the formation of inclusion bodies that were subsequently denatured by treatment with urea and allowed to refoldxa0in vitro. Activity assays of the recombinant Afp1 confirmed the antifreeze protein properties with a high TH value of 0.08xa0°C.

Cytotoxic Activities of Major Diterpenoid Constituents of Andrographis paniculata. in a Panel of Human Tumor Cell Lines

ABSTRACT The cytotoxic activities of several diterpenoid constituents of the medicinal plant, “hempedu bumi,” or Andrographis paniculata. Nees (Acanthaceae), were evaluated. The seven diterpenoid compounds used were andrographolide, 14-deoxyandrographolide, andrographiside, deoxyandrographiside, 14-deoxy-12-methoxyandrographolide, neoandrographolide, and 14-deoxy-11,12-didehydroandrographolide. The activities of these compounds were evaluated with various human tumor cell lines such as Caov-3 (human ovarian carcinoma cell line), T-47D (human breast carcinoma cell line), Hs-578T (human breast carcinoma cell line), Hep G2 (human hepatocarcinoma cell line), and NCI–H23 (human lung adenocarcinoma cell line). Cell survival was measured using the MTS assay after 72 h of incubation. Andrographolide, neoandrographolide, andrographiside, deoxyandrographiside, and 14-deoxy-12-methoxyandrographolide appeared to be noncytotoxic against all the cell lines. Only 14-deoxyandrographolide and 14-deoxy-11,12-didehydroandrographolide exhibited cytotoxic activities (based on EC50 values), but this was limited to the T-47D cell line (EC50 values of 2.8 µg/ml and 1.5 µg/ml, respectively). As one of the principle diterpenoids of Andrographis paniculata., 14-deoxy-11,12-didehydroandrographolide appeared to be the most potent when compared with the rest of the compounds examined. The effects of 14-deoxy-11,12-didehydroandrographolide on T-47D cells were further confirmed to be nonapoptotic, non-necrotic, but programmed in nature, as demonstrated using a DNA fragmentation detection assay, Trypan blue exclusion assay, and annexin V–propidium iodide staining.

Molecular characterisation of six alternatively spliced variants and a novel promoter in human peroxisome proliferator-activated receptor α

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a ligand-activated transcriptional factor that governs many biological processes, including lipid metabolism, inflammation, and atherosclerosis. We demonstrate here the existence of six variants and multiple transcriptional start sites of the 5() untranslated region (UTR) of hPPARalpha gene, originating from the use of alternative splicing mechanisms and four different promoters. Three new novel exons at the 5()-untranslated region of human PPARalpha gene were also identified and designated as Exon A, Exon B, and Exon 2b. In addition, 1.2kb promoter fragment which drives the transcription of 2 variants with Exon B (hPPARalpha4 and 6) was successfully cloned and characterised. Sequencing results revealed promoter B did not contain a conservative TATA box within the first 100 nucleotides from transcriptional start site but has several GC-rich regions and putative Sp1 sites. Using luciferase reporter constructs transfected into HepG2 and Hep3B cell lines, promoter B was shown to be functionally active. Basal transcriptional activity was significantly high in the promoter fragment -341/+34, but lower in the region -341/-1147 as compared to the fragment -341/+34, indicating the presence of an element conferring transcriptional activation between positions -341 and +34 or alternatively, the presence of transcriptional repression between positions -341 and -1147 in the promoter B of hPPARalpha.

Hell's Gate globin I: An acid and thermostable bacterial hemoglobin resembling mammalian neuroglobin

Hells Gate globin I (HGbI), a heme‐containing protein structurally homologous to mammalian neuroglobins, has been identified from an acidophilic and thermophilic obligate methanotroph, Methylacidiphilum infernorum. HGbI has very high affinity for O2 and shows barely detectable autoxidation in the pH range of 5.2–8.6 and temperature range of 25–50 °C. Examination of the heme pocket by X‐ray crystallography and molecular dynamics showed that conformational movements of Tyr29(B10) and Gln50(E7), as well as structural flexibility of the GH loop and H‐helix, may play a role in modulating its ligand binding behavior. Bacterial HGbIs unique resistance to the sort of extreme acidity that would extract heme from any other hemoglobin makes it an ideal candidate for comparative structure–function studies of the expanding globin superfamily.

Site-directed saturation mutagenesis at residue F420 and recombination with another beneficial mutation of Ralstonia eutropha polyhydroxyalkanoate synthase.

The F420S substitution enhances the specific activity of Ralstonia eutropha PHA synthase (PhaCRe). We have now carried out site-directed saturation mutagenesis of F420 of PhaCRe and, amongst the F420 mutants, the F420S mutant gave the highest poly(3-hydroxybutyrate) (PHB) content. In vitro activity assay showed that the F420S enzyme had a significant decrease in its lag phase compared to that of the wild-type enzyme. Enhancement of PHB accumulation was achieved by combination of the F420S mutation with a G4D mutation, which conferred high PHB content and high in vivo concentration of PhaCRe enzyme. The G4D/F420S mutant gave a higher PHB content and in vivo concentration of PhaCRe enzyme than the F420S mutant, while the molecular weight of the PHB polymer of the double mutant was similar to that of the F420S mutant.

Crystal structure of a compact α-amylase from Geobacillus thermoleovorans.

A truncated form of an α-amylase, GTA, from thermophilic Geobacillus thermoleovorans CCB_US3_UF5 was biochemically and structurally characterized. The recombinant GTA, which lacked both the N- and C-terminal transmembrane regions, functioned optimally at 70°C and pH 6.0. While enzyme activity was not enhanced by the addition of CaCl2, GTAs thermostability was significantly improved in the presence of CaCl2. The structure, in complex with an acarbose-derived pseudo-hexasaccharide, consists of the typical three domains and binds one Ca(2+) ion. This Ca(2+) ion was strongly bound and not chelated by EDTA. A predicted second Ca(2+)-binding site, however, was disordered. With limited subsites, two novel substrate-binding residues, Y147 and Y182, may help increase substrate affinity. No distinct starch-binding domain is present, although two regions rich in aromatic residues have been observed. GTA, with a smaller domain B and several shorter loops compared to other α-amylases, has one of the most compact α-amylase folds that may contribute greatly to its tight Ca(2+) binding and thermostability.