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1.  Role of Key Salt Bridges in Thermostability of G. thermodenitrificans EstGtA2: Distinctive Patterns within the New Bacterial Lipolytic Enzyme Family XV 
PLoS ONE  2013;8(10):e76675.
Bacterial lipolytic enzymes were originally classified into eight different families defined by Arpigny and Jaeger (families I-VIII). Recently, the discovery of new lipolytic enzymes allowed for extending the original classification to fourteen families (I-XIV). We previously reported that G. thermodenitrificans EstGtA2 (access no. AEN92268) belonged to a novel group of bacterial lipolytic enzymes. Here we propose a 15th family (family XV) and suggest criteria for the assignation of protein sequences to the N’ subfamily. Five selected salt bridges, hallmarks of the N’ subfamily (E3/R54, E12/R37, E66/R140, D124/K178 and D205/R220) were disrupted in EstGtA2 using a combinatorial alanine-scanning approach. A set of 14 (R/K→A) mutants was produced, including five single, three double, three triple and three quadruple mutants. Despite a high tolerance to non-conservative mutations for folding, all the alanine substitutions were destabilizing (decreasing Tm by 5 to 14°C). A particular combination of four substitutions exceeded this tolerance and prevents the correct folding of EstGtA2, leading to enzyme inactivation. Although other mutants remain active at low temperatures, the accumulation of more than two mutations had a dramatic impact on EstGtA2 activity at high temperatures suggesting an important role of these conserved salt bridge-forming residues in thermostability of lipolytic enzymes from the N’ subfamily. We also identified a particular interloop salt bridge in EstGtA2 (D194/H222), located at position i -2 and i -4 residues from the catalytic Asp and His respectively which is conserved in other related bacterial lipolytic enzymes (families IV and XIII) with high tolerance to mutations and charge reversal. We investigated the role of residue identity at position 222 in controlling stability-pH dependence in EstGtA2. The introduction of a His to Arg mutation led to increase thermostability under alkaline pH. Our results suggest primary targets for optimization of EstGtA2 for specific biotechnological purposes.
PMCID: PMC3792869  PMID: 24116134
2.  Dieselzymes: development of a stable and methanol tolerant lipase for biodiesel production by directed evolution 
Biodiesels are methyl esters of fatty acids that are usually produced by base catalyzed transesterification of triacylglyerol with methanol. Some lipase enzymes are effective catalysts for biodiesel synthesis and have many potential advantages over traditional base or acid catalyzed transesterification. Natural lipases are often rapidly inactivated by the high methanol concentrations used for biodiesel synthesis, however, limiting their practical use. The lipase from Proteus mirabilis is a particularly promising catalyst for biodiesel synthesis as it produces high yields of methyl esters even in the presence of large amounts of water and expresses very well in Escherichia coli. However, since the Proteus mirabilis lipase is only moderately stable and methanol tolerant, these properties need to be improved before the enzyme can be used industrially.
We employed directed evolution, resulting in a Proteus mirabilis lipase variant with 13 mutations, which we call Dieselzyme 4. Dieselzyme 4 has greatly improved thermal stability, with a 30-fold increase in the half-inactivation time at 50°C relative to the wild-type enzyme. The evolved enzyme also has dramatically increased methanol tolerance, showing a 50-fold longer half-inactivation time in 50% aqueous methanol. The immobilized Dieselzyme 4 enzyme retains the ability to synthesize biodiesel and has improved longevity over wild-type or the industrially used Brukholderia cepacia lipase during many cycles of biodiesel synthesis. A crystal structure of Dieselzyme 4 reveals additional hydrogen bonds and salt bridges in Dieselzyme 4 compared to the wild-type enzyme, suggesting that polar interactions may become particularly stabilizing in the reduced dielectric environment of the oil and methanol mixture used for biodiesel synthesis.
Directed evolution was used to produce a stable lipase, Dieselzyme 4, which could be immobilized and re-used for biodiesel synthesis. Dieselzyme 4 outperforms the industrially used lipase from Burkholderia cepacia and provides a platform for still further evolution of desirable biodiesel production properties.
PMCID: PMC3670234  PMID: 23648063
Lipase; Biodiesel; Proteus mirabilis; Proteus sp. K107; Directed evolution; Alcohol tolerance
3.  Identification of Thermophilic Bacterial Strains Producing Thermotolerant Hydrolytic Enzymes from Manure Compost 
Indian Journal of Microbiology  2011;52(1):41-47.
Ten thermophilic bacterial strains were isolated from manure compost. Phylogenetic analysis based on 16S rRNA genes and biochemical characterization allowed identification of four different species belonging to four genera: Geobacillus thermodenitrificans, Bacillus smithii, Ureibacillus suwonensis and Aneurinibacillus thermoaerophilus. PCR-RFLP profiles of the 16S-ITS-23S rRNA region allowed us to distinguish two subgroups among the G. thermodenitrificans isolates. Isolates were screened for thermotolerant hydrolytic activities (60–65°C). Thermotolerant lipolytic activities were detected for G. thermodenitrificans, A. thermoaerophilus and B. smithii. Thermotolerant protease, α-amylase and xylanase activities were also observed in the G. thermodenitrificans group. These species represent a source of potential novel thermostable enzymes for industrial applications.
PMCID: PMC3298584  PMID: 23448754
Manure compost; Thermophilic bacteria; 16S rDNA; Hydrolytic enzymes; Thermal stability
4.  Earth as an Extrasolar Planet: Earth Model Validation Using EPOXI Earth Observations 
Astrobiology  2011;11(5):393-408.
The EPOXI Discovery Mission of Opportunity reused the Deep Impact flyby spacecraft to obtain spatially and temporally resolved visible photometric and moderate resolution near-infrared (NIR) spectroscopic observations of Earth. These remote observations provide a rigorous validation of whole-disk Earth model simulations used to better understand remotely detectable extrasolar planet characteristics. We have used these data to upgrade, correct, and validate the NASA Astrobiology Institute's Virtual Planetary Laboratory three-dimensional line-by-line, multiple-scattering spectral Earth model. This comprehensive model now includes specular reflectance from the ocean and explicitly includes atmospheric effects such as Rayleigh scattering, gas absorption, and temperature structure. We have used this model to generate spatially and temporally resolved synthetic spectra and images of Earth for the dates of EPOXI observation. Model parameters were varied to yield an optimum fit to the data. We found that a minimum spatial resolution of ∼100 pixels on the visible disk, and four categories of water clouds, which were defined by using observed cloud positions and optical thicknesses, were needed to yield acceptable fits. The validated model provides a simultaneous fit to Earth's lightcurve, absolute brightness, and spectral data, with a root-mean-square (RMS) error of typically less than 3% for the multiwavelength lightcurves and residuals of ∼10% for the absolute brightness throughout the visible and NIR spectral range. We have extended our validation into the mid-infrared by comparing the model to high spectral resolution observations of Earth from the Atmospheric Infrared Sounder, obtaining a fit with residuals of ∼7% and brightness temperature errors of less than 1 K in the atmospheric window. For the purpose of understanding the observable characteristics of the distant Earth at arbitrary viewing geometry and observing cadence, our validated forward model can be used to simulate Earth's time-dependent brightness and spectral properties for wavelengths from the far ultraviolet to the far infrared. Key Words: Astrobiology—Extrasolar terrestrial planets—Habitability—Planetary science—Radiative transfer. Astrobiology 11, 393–408.
PMCID: PMC3133830  PMID: 21631250
5.  ACE Genotype and the Muscle Hypertrophic and Strength Responses to Strength Training 
Previous studies have linked an insertion/deletion polymorphism in the angiotensin-converting enzyme (ACE) gene with variability in muscle strength responses to strength training (ST), though conclusions have been inconsistent across investigations. Moreover, most previous studies have not investigated the influence of sex on the association of ACE I/D genotype with muscle phenotypes. The purpose of this study was to investigate the association of ACE genotype with muscle phenotypes before and after ST in older men and women.
Eighty-six inactive men and 139 inactive women, ages 50–85 yr (mean: 62 yr), were studied before and after 10 wk of unilateral knee extensor ST. The one-repetition maximum (1RM) test was used to assess knee extensor muscle strength, and computed tomography was used to measure quadriceps muscle volume (MV). Differences were compared among ACE genotype groups (II vs ID vs DD).
Across the entire cohort at baseline, ACE genotype was significantly associated with total lean mass and body weight, with higher values in DD genotype carriers (both P < 0.05). At baseline, DD genotype carriers exhibited significantly greater MV compared with II genotype carriers for both the trained leg (men: 1828 ± 44 vs 1629 ± 70; women: 1299 ± 34 vs 1233 ± 49; P = 0.02) and untrained leg (men: 1801 ± 46 vs 1559 ± 72; women: 1268 ± 36 vs 1189 ± 51; P = 0.01), with no significant genotype × sex interaction. No ACE genotype associations were observed for the 1RM or MV adaptations to ST in either men or women.
In the present study, ACE genotype was associated with baseline differences in muscle volume, but it was not associated with the muscle hypertrophic response to ST.
PMCID: PMC2984550  PMID: 18317377
6.  Structural characterization of cationic lipid–tRNA complexes 
Nucleic Acids Research  2009;37(15):5197-5207.
Despite considerable interest and investigations on cationic lipid–DNA complexes, reports on lipid–RNA interaction are very limited. In contrast to lipid–DNA complexes where lipid binding induces partial B to A and B to C conformational changes, lipid–tRNA complexation preserves tRNA folded state. This study is the first attempt to investigate the binding of cationic lipid with transfer RNA and the effect of lipid complexation on tRNA aggregation and condensation. We examine the interaction of tRNA with cholesterol (Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethylammoniumbromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE), at physiological condition, using constant tRNA concentration and various lipid contents. FTIR, UV-visible, CD spectroscopic methods and atomic force microscopy (AFM) were used to analyze lipid binding site, the binding constant and the effects of lipid interaction on tRNA stability, conformation and condensation. Structural analysis showed lipid–tRNA interactions with G–C and A–U base pairs as well as the backbone phosphate group with overall binding constants of KChol = 5.94 (± 0.8) × 104 M–1, KDDAB = 8.33 (± 0.90) × 105 M–1, KDOTAP = 1.05 (± 0.30) × 105 M–1 and KDOPE = 2.75 (± 0.50) × 104 M–1. The order of stability of lipid–tRNA complexation is DDAB > DOTAP > Chol > DOPE. Hydrophobic interactions between lipid aliphatic tails and tRNA were observed. RNA remains in A-family structure, while biopolymer aggregation and condensation occurred at high lipid concentrations.
PMCID: PMC2731917  PMID: 19561199
7.  Structural analysis of DNA complexation with cationic lipids 
Nucleic Acids Research  2008;37(3):849-857.
Complexes of cationic liposomes with DNA are promising tools to deliver genetic information into cells for gene therapy and vaccines. Electrostatic interaction is thought to be the major force in lipid–DNA interaction, while lipid-base binding and the stability of cationic lipid–DNA complexes have been the subject of more debate in recent years. The aim of this study was to examine the complexation of calf-thymus DNA with cholesterol (Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethylammoniumbromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE), at physiological condition, using constant DNA concentration and various lipid contents. Fourier transform infrared (FTIR), UV-visible, circular dichroism spectroscopic methods and atomic force microscopy were used to analyse lipid-binding site, the binding constant and the effects of lipid interaction on DNA stability and conformation. Structural analysis showed a strong lipid–DNA interaction via major and minor grooves and the backbone phosphate group with overall binding constants of KChol = 1.4 (±0.5) × 104 M−1, KDDAB = 2.4 (±0.80) × 104 M−1, KDOTAP = 3.1 (±0.90) × 104 M−1 and KDOPE = 1.45 (± 0.60) × 104 M−1. The order of stability of lipid–DNA complexation is DOTAP>DDAB>DOPE>Chol. Hydrophobic interactions between lipid aliphatic tails and DNA were observed. Chol and DOPE induced a partial B to A-DNA conformational transition, while a partial B to C-DNA alteration occurred for DDAB and DOTAP at high lipid concentrations. DNA aggregation was observed at high lipid content.
PMCID: PMC2647290  PMID: 19103664

Results 1-7 (7)