-
Protein Science : a Publication of the... Aug 2006In this work we compare the dynamics and conformational stability of Pseudomonas mendocina lipase enzyme and its F180P/S205G mutant that shows higher activity and... (Comparative Study)
Comparative Study
In this work we compare the dynamics and conformational stability of Pseudomonas mendocina lipase enzyme and its F180P/S205G mutant that shows higher activity and stability for use in washing powders. Our NMR analyses indicate virtually identical structures but reveal remarkable differences in local dynamics, with striking correspondence between experimental data (i.e., (15)N relaxation and H/D exchange rates) and data from Molecular Dynamics simulations. While overall the cores of both proteins are very rigid on the pico- to nanosecond timescale and are largely protected from H/D exchange, the two point mutations stabilize helices alpha1, alpha4, and alpha5 and locally destabilize the H-bond network of the beta-sheet (beta7-beta9). In particular, it emerges that helix alpha5, undergoing some fast destabilizing motions (on the pico- to nanosecond timescale) in wild-type lipase, is substantially rigidified by the mutation of Phe180 for a proline at its N terminus. This observation could be explained by the release of some penalizing strain, as proline does not require any "N-capping" hydrogen bond acceptor in the i+3 position. The combined experimental and simulated data thus indicate that reduced molecular flexibility of the F180P/S205G mutant lipase underlies its increased stability, and thus reveals a correlation between microscopic dynamics and macroscopic thermodynamic properties. This could contribute to the observed altered enzyme activity, as may be inferred from recent studies linking enzyme kinetics to their local molecular dynamics.
Topics: Amino Acid Sequence; Deuterium Exchange Measurement; Enzyme Stability; Hot Temperature; Hydrogen Bonding; Lipase; Nuclear Magnetic Resonance, Biomolecular; Point Mutation; Protein Conformation; Protein Denaturation; Protein Structure, Secondary; Pseudomonas mendocina; Urea
PubMed: 16823035
DOI: 10.1110/ps.062213706 -
Plant Disease Apr 2006A soil inoculant, Vesta (Biologically Integrated Organics, Inc., Sonoma, CA), was tested for its ability to inhibit Armillaria mellea, causal agent of Armillaria root...
A soil inoculant, Vesta (Biologically Integrated Organics, Inc., Sonoma, CA), was tested for its ability to inhibit Armillaria mellea, causal agent of Armillaria root disease of grapevine (Vitis vinifera). Colony diameter of A. mellea was significantly inhibited by undiluted inoculant (P < 0.0001) and by bacterial isolates cultured from the inoculant (Bacillus subtilis, B. lentimorbus, Comamonas testosteroni, Pseudomonas aeruginosa, P. mendocina; P < 0.0001) relative to diameter of the nontreated control. Efficacy of the inoculant for postinfection control of Armillaria root disease of grapevine was examined in an A. mellea-infested vineyard in northern California. Inoculant was applied via drip-irrigation to vine rows in replicate blocks in 2003 and 2004. Yield, growth, mineral nutrition, and juice quality parameters of healthy and symptomatic vines were measured in treated and nontreated vine rows. Significantly decreased petiole P and K concentrations and significantly lower soluble solids content in fruit from symptomatic vines demonstrated that Armillaria root disease negatively affects vine mineral nutritional status and fruit quality, findings that have not been previously reported for an agronomic host of A. mellea. The inoculant significantly increased cluster weights of symptomatic vines (109.63 g/cluster), relative to those of symptomatic-nontreated vines (92.05 g/cluster), to levels comparable to those of healthy vines (122.09 g/cluster). However, the inoculant did not decrease the rate of symptom development or mortality of treated vines from 2002 to 2004. The results of our field experiment suggest that the inoculant may not prevent Armillaria root disease, but can provide therapeutic benefit by improving productivity of infected vines.
PubMed: 30786591
DOI: 10.1094/PD-90-0439 -
FEMS Microbiology Ecology Feb 2005Denaturing gradient gel electrophoresis of amplified fragments of genes coding for 16S rRNA and for the largest subunit of multicomponent phenol hydroxylase (LmPH) was...
Denaturing gradient gel electrophoresis of amplified fragments of genes coding for 16S rRNA and for the largest subunit of multicomponent phenol hydroxylase (LmPH) was used to monitor the behaviour and relative abundance of mixed phenol-degrading bacterial populations (Pseudomonas mendocina PC1, P. fluorescens strains PC18, PC20 and PC24) during degradation of phenolic compounds in phenolic leachate- and oil-amended microcosms. The analysis indicated that specific bacterial populations were selected in each microcosm. The naphthalene-degrading strain PC20 was the dominant degrader in oil-amended microcosms and strain PC1 in phenolic leachate microcosms. Strain PC20 was not detectable after cultivation in phenolic leachate microcosms. Mixed bacterial populations in oil-amended microcosms aggregated and formed clumps, whereas the same bacteria had a planktonic mode of growth in phenolic leachate microcosms. Colony hybridisation data with catabolic gene specific probes indicated that, in leachate microcosms, the relative proportions of bacteria having meta (PC1) and ortho (PC24) pathways for degradation of phenol and p-cresol changed alternately. The shifts in the composition of mixed population indicated that different pathways of metabolism of aromatic compounds dominated and that this process is an optimised response to the contaminants present in microcosms.
Topics: Biodegradation, Environmental; Culture Media; DNA, Bacterial; DNA, Ribosomal; Ecosystem; Electrophoresis; Mixed Function Oxygenases; Molecular Sequence Data; Petroleum; Phenols; Plankton; Polymerase Chain Reaction; Pseudomonas; Pseudomonas fluorescens; RNA, Ribosomal, 16S
PubMed: 16329884
DOI: 10.1016/j.femsec.2004.09.009 -
TheScientificWorldJournal Jul 2005Collections of native Pseudomonas spp. are kept at the NCAM of Uzbekistan. Some of those organisms were isolated from the rhizosphere of cotton, wheat, corn, melon,...
Collections of native Pseudomonas spp. are kept at the NCAM of Uzbekistan. Some of those organisms were isolated from the rhizosphere of cotton, wheat, corn, melon, alfalfa, and tomato grown in field locations within a semi-arid region of Uzbekistan. Strains used for this study were Pseudomonas alcaligenes, P. aurantiaca, P. aureofaciens, P. denitrificans, P. mendocina, P. rathonis, and P. stutzeri. Some of the pseudomonads have been characterized in this report. These strains produced enzymes, phytohormone auxin (IAA), and were antagonist against plant pathogenic fungi in in vitro experiments. Most of the strains were salt tolerant and temperature resistant. Some of the Pseudomonas spp. isolated in this study have been found to increase the growth of wheat, corn, and tomato in pot experiments.
Topics: Carbohydrate Metabolism; Climate; Ecosystem; Fermentation; Fungi; Plant Development; Plant Roots; Plant Shoots; Plants; Pseudomonas; Soil Microbiology; Uzbekistan
PubMed: 16075145
DOI: 10.1100/tsw.2005.64 -
Applied and Environmental Microbiology Jul 2005Methyl bromide (CH3Br) and methyl chloride (CH3Cl) are important precursors for destruction of stratospheric ozone, and oceanic uptake is an important component of the...
Methyl bromide (CH3Br) and methyl chloride (CH3Cl) are important precursors for destruction of stratospheric ozone, and oceanic uptake is an important component of the biogeochemical cycle of these methyl halides. In an effort to identify and characterize the organisms mediating halocarbon biodegradation, we surveyed the effect of potential cometabolic substrates on CH3Br biodegradation using a 13CH3Br incubation technique. Toluene (160 to 200 nM) clearly inhibited CH3Br and CH3Cl degradation in seawater samples from the North Atlantic, North Pacific, and Southern Oceans. Furthermore, a marine bacterium able to co-oxidize CH3Br while growing on toluene was isolated from subtropical Western Atlantic seawater. The bacterium, Oxy6, was also able to oxidize o-xylene and the xylene monooxygenase (XMO) pathway intermediate 3-methylcatechol. Patterns of substrate oxidation, lack of acetylene inhibition, and the inability of the toluene 4-monooxygenase (T4MO)-containing bacterium Pseudomonas mendocina KR1 to degrade CH3Br ruled out participation of the T4MO pathway in Oxy6. Oxy6 also oxidized a variety of toluene (TOL) pathway intermediates such as benzyl alcohol, benzylaldehyde, benzoate, and catechol, but the inability of Pseudomonas putida mt-2 to degrade CH3Br suggested that the TOL pathway might not be responsible for CH3Br biodegradation. Molecular phylogenetic analysis identified Oxy6 to be a member of the family Sphingomonadaceae related to species within the Porphyrobacter genus. Although some Sphingomonadaceae can degrade a variety of xenobiotic compounds, this appears to be the first report of CH3Br degradation for this class of organism. The widespread inhibitory effect of toluene on natural seawater samples and the metabolic capabilities of Oxy6 indicate a possible link between aromatic hydrocarbon utilization and the biogeochemical cycle of methyl halides.
Topics: Biodegradation, Environmental; Carbon Isotopes; Culture Media; Hydrocarbons, Brominated; Methyl Chloride; Molecular Sequence Data; Oxidation-Reduction; Phylogeny; Seawater; Sequence Analysis, DNA; Sphingomonadaceae; Toluene
PubMed: 16000753
DOI: 10.1128/AEM.71.7.3495-3503.2005 -
Journal of Bacteriology Aug 2004A combined phylogenetic and multilocus DNA sequence analysis of 26 Pseudomonas stutzeri strains distributed within the 9 genomovars of the species has been performed.... (Comparative Study)
Comparative Study
A combined phylogenetic and multilocus DNA sequence analysis of 26 Pseudomonas stutzeri strains distributed within the 9 genomovars of the species has been performed. Type strains of the two most closely related species (P. balearica, former genomovar 6, and P. mendocina), together with P. aeruginosa, as the type species of the genus, have been included in the study. The extremely high genetic diversity and the clonal structure of the species were confirmed by the sequence analysis. Clustering of strains in the consensus phylogeny inferred from the analysis of seven nucleotide sequences (16S ribosomal DNA, internally transcribed spacer region 1, gyrB, rpoD, nosZ, catA, and nahH) confirmed the monophyletic origin of the genomovars within the Pseudomonas branch and is in good agreement with earlier DNA-DNA similarity analysis, indicating that the selected genes are representative of the whole genome in members of the species.
Topics: Alleles; Bacterial Proteins; DNA Gyrase; DNA, Bacterial; DNA-Directed RNA Polymerases; Genes, Bacterial; Genetic Variation; Genotype; Molecular Sequence Data; Phylogeny; Pseudomonas aeruginosa; Pseudomonas mendocina; Pseudomonas stutzeri; RNA, Bacterial; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Sequence Homology; Sigma Factor; Transcription Factors
PubMed: 15292125
DOI: 10.1128/JB.186.16.5239-5248.2004 -
Applied and Environmental Microbiology Jul 2004Aromatic hydroxylations are important bacterial metabolic processes but are difficult to perform using traditional chemical synthesis, so to use a biological catalyst to...
Oxidation of benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by toluene 4-monooxygenase of Pseudomonas mendocina KR1 and toluene 3-monooxygenase of Ralstonia pickettii PKO1.
Aromatic hydroxylations are important bacterial metabolic processes but are difficult to perform using traditional chemical synthesis, so to use a biological catalyst to convert the priority pollutant benzene into industrially relevant intermediates, benzene oxidation was investigated. It was discovered that toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1, toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1, and toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 convert benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by successive hydroxylations. At a concentration of 165 microM and under the control of a constitutive lac promoter, Escherichia coli TG1/pBS(Kan)T4MO expressing T4MO formed phenol from benzene at 19 +/- 1.6 nmol/min/mg of protein, catechol from phenol at 13.6 +/- 0.3 nmol/min/mg of protein, and 1,2,3-trihydroxybenzene from catechol at 2.5 +/- 0.5nmol/min/mg of protein. The catechol and 1,2,3-trihydroxybenzene products were identified by both high-pressure liquid chromatography and mass spectrometry. When analogous plasmid constructs were used, E. coli TG1/pBS(Kan)T3MO expressing T3MO formed phenol, catechol, and 1,2,3-trihydroxybenzene at rates of 3 +/- 1, 3.1 +/- 0.3, and 0.26 +/- 0.09 nmol/min/mg of protein, respectively, and E. coli TG1/pBS(Kan)TOM expressing TOM formed 1,2,3-trihydroxybenzene at a rate of 1.7 +/- 0.3 nmol/min/mg of protein (phenol and catechol formation rates were 0.89 +/- 0.07 and 1.5 +/- 0.3 nmol/min/mg of protein, respectively). Hence, the rates of synthesis of catechol by both T3MO and T4MO and the 1,2,3-trihydroxybenzene formation rate by TOM were found to be comparable to the rates of oxidation of the natural substrate toluene for these enzymes (10.0 +/- 0.8, 4.0 +/- 0.6, and 2.4 +/- 0.3 nmol/min/mg of protein for T4MO, T3MO, and TOM, respectively, at a toluene concentration of 165 microM).
Topics: Base Sequence; Benzene; Catechols; DNA, Bacterial; Molecular Sequence Data; Oxidation-Reduction; Oxygenases; Phenol; Pseudomonas mendocina; Pyrogallol; Ralstonia
PubMed: 15240250
DOI: 10.1128/AEM.70.7.3814-3820.2004 -
Journal of Bacteriology Jul 2004Wild-type toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 oxidizes toluene to p-cresol (96%) and oxidizes benzene sequentially to phenol, to catechol, and to...
Wild-type toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 oxidizes toluene to p-cresol (96%) and oxidizes benzene sequentially to phenol, to catechol, and to 1,2,3-trihydroxybenzene. In this study T4MO was found to oxidize o-cresol to 3-methylcatechol (91%) and methylhydroquinone (9%), to oxidize m-cresol and p-cresol to 4-methylcatechol (100%), and to oxidize o-methoxyphenol to 4-methoxyresorcinol (87%), 3-methoxycatechol (11%), and methoxyhydroquinone (2%). Apparent Vmax values of 6.6 +/- 0.9 to 10.7 +/- 0.1 nmol/min/ mg of protein were obtained for o-, m-, and p-cresol oxidation by wild-type T4MO, which are comparable to the toluene oxidation rate (15.1 +/- 0.8 nmol/min/mg of protein). After these new reactions were discovered, saturation mutagenesis was performed near the diiron catalytic center at positions I100, G103, and A107 of the alpha subunit of the hydroxylase (TmoA) based on directed evolution of the related toluene o-monooxygenase of Burkholderia cepacia G4 (K. A. Canada, S. Iwashita, H. Shim, and T. K. Wood, J. Bacteriol. 184:344-349, 2002) and a previously reported T4MO G103L regiospecific mutant (K. H. Mitchell, J. M. Studts, and B. G. Fox, Biochemistry 41:3176-3188, 2002). By using o-cresol and o-methoxyphenol as model substrates, regiospecific mutants of T4MO were created; for example, TmoA variant G103A/A107S produced 3-methylcatechol (98%) from o-cresol twofold faster and produced 3-methoxycatechol (82%) from 1 mM o-methoxyphenol seven times faster than the wild-type T4MO (1.5 +/- 0.2 versus 0.21 +/- 0.01 nmol/min/mg of protein). Variant I100L produced 3-methoxycatechol from o-methoxyphenol four times faster than wild-type T4MO, and G103S/A107T produced methylhydroquinone (92%) from o-cresol fourfold faster than wild-type T4MO and there was 10 times more in terms of the percentage of the product. Variant G103S produced 40-fold more methoxyhydroquinone from o-methoxyphenol than the wild-type enzyme produced (80 versus 2%) and produced methylhydroquinone (80%) from o-cresol. Hence, the regiospecific oxidation of o-methoxyphenol and o-cresol was changed for significant synthesis of 3-methoxycatechol, methoxyhydroquinone, 3-methylcatechol, and methylhydroquinone. The enzyme variants also demonstrated altered monohydroxylation regiospecificity for toluene; for example, G103S/A107G formed 82% o-cresol, so saturation mutagenesis converted T4MO into an ortho-hydroxylating enzyme. Furthermore, G103S/A107T formed 100% p-cresol from toluene; hence, a better para-hydroxylating enzyme than wild-type T4MO was formed. Structure homology modeling suggested that hydrogen bonding interactions of the hydroxyl groups of altered residues S103, S107, and T107 influence the regiospecificity of the oxygenase reaction.
Topics: Amino Acid Substitution; Benzene; Binding Sites; Catechols; Cresols; Hydroquinones; Models, Molecular; Mutagenesis; Oxidation-Reduction; Oxygenases; Phenol; Protein Subunits; Pseudomonas mendocina; Pyrogallol; Quantitative Structure-Activity Relationship; Substrate Specificity; Toluene
PubMed: 15231803
DOI: 10.1128/JB.186.14.4705-4713.2004 -
Journal of Bacteriology May 2004Oxygenases are promising biocatalysts for performing selective hydroxylations not accessible by chemical methods. Whereas toluene 4-monooxygenase (T4MO) of Pseudomonas...
Oxygenases are promising biocatalysts for performing selective hydroxylations not accessible by chemical methods. Whereas toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 hydroxylates monosubstituted benzenes at the para position and toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 hydroxylates at the ortho position, toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1 was reported previously to hydroxylate toluene at the meta position, producing primarily m-cresol (R. H. Olsen, J. J. Kukor, and B. Kaphammer, J. Bacteriol. 176:3749-3756, 1994). Using gas chromatography, we have discovered that T3MO hydroxylates monosubstituted benzenes predominantly at the para position. TG1/pBS(Kan)T3MO cells expressing T3MO oxidized toluene at a maximal rate of 11.5 +/- 0.33 nmol/min/mg of protein with an apparent Km value of 250 microM and produced 90% p-cresol and 10% m-cresol. This product mixture was successively transformed to 4-methylcatechol. T4MO, in comparison, produces 97% p-cresol and 3% m-cresol. Pseudomonas aeruginosa PAO1 harboring pRO1966 (the original T3MO-bearing plasmid) also exhibited the same product distribution as that of TG1/pBS(Kan)T3MO. TG1/pBS(Kan)T3MO produced 66% p-nitrophenol and 34% m-nitrophenol from nitrobenzene and 100% p-methoxyphenol from methoxybenzene, as well as 62% 1-naphthol and 38% 2-naphthol from naphthalene; similar results were found with TG1/pBS(Kan)T4MO. Sequencing of the tbu locus from pBS(Kan)T3MO and pRO1966 revealed complete identity between the two, thus eliminating any possible cloning errors. 1H nuclear magnetic resonance analysis confirmed the structural identity of p-cresol in samples containing the product of hydroxylation of toluene by pBS(Kan)T3MO.
Topics: Hydroxylation; Magnetic Resonance Spectroscopy; Oxidation-Reduction; Oxygenases; Ralstonia; Toluene
PubMed: 15126473
DOI: 10.1128/JB.186.10.3117-3123.2004 -
Applied and Environmental Microbiology Dec 2003Pseudomonas mendocina KR-1 grew well on toluene, n-alkanes (C5 to C8), and 1 degrees alcohols (C2 to C8) but not on other aromatics, gaseous n-alkanes (C1 to C4),...
Pseudomonas mendocina KR-1 grew well on toluene, n-alkanes (C5 to C8), and 1 degrees alcohols (C2 to C8) but not on other aromatics, gaseous n-alkanes (C1 to C4), isoalkanes (C4 to C6), 2 degrees alcohols (C3 to C8), methyl tertiary butyl ether (MTBE), or tertiary butyl alcohol (TBA). Cells grown under carbon-limited conditions on n-alkanes in the presence of MTBE (42 micromoles) oxidized up to 94% of the added MTBE to TBA. Less than 3% of the added MTBE was oxidized to TBA when cells were grown on either 1 degrees alcohols, toluene, or dextrose in the presence of MTBE. Concentrated n-pentane-grown cells oxidized MTBE to TBA without a lag phase and without generating tertiary butyl formate (TBF) as an intermediate. Neither TBF nor TBA was consumed by n-pentane-grown cells, while formaldehyde, the expected C1 product of MTBE dealkylation, was rapidly consumed. Similar Ks values for MTBE were observed for cells grown on C5 to C8 n-alkanes (12.95 +/- 2.04 mM), suggesting that the same enzyme oxidizes MTBE in cells grown on each n-alkane. All growth-supporting n-alkanes (C5 to C8) inhibited MTBE oxidation by resting n-pentane-grown cells. Propane (Ki = 53 micromoles) and n-butane (Ki = 16 micromoles) also inhibited MTBE oxidation, and both gases were also consumed by cells during growth on n-pentane. Cultures grown on C5 to C8 n-alkanes also exhibited up to twofold-higher levels of growth in the presence of propane or n-butane, whereas no growth stimulation was observed with methane, ethane, MTBE, TBA, or formaldehyde. The results are discussed in terms of their impacts on our understanding of MTBE biodegradation and cometabolism.
Topics: Alkanes; Culture Media; Kinetics; Methyl Ethers; Oxidation-Reduction; Pseudomonas mendocina; Time Factors
PubMed: 14660389
DOI: 10.1128/AEM.69.12.7385-7394.2003