High yield production in seven days of Coriolopsis gallica 1184 laccase at 50 L scale; enzyme purification and molecular characterization.

The optimization of culture conditions for high yield laccase production by white rot fungi has been extensively studied. However, to achieve short time laccase production remains a major challenge in several cases. The present study investigated an optimal process for production of Coriolopsis gallica 1184 laccase in a high yield of 200 900 Ul(-1) in 7 d by 50 L scale submerged fermentation. Coriolopsis gallica 1184 laccase appeared as a robust enzyme against downstream process; only 13.5 % of laccase activity was lost at the end of downstream procedure. The pure enzyme appeared as a one-species laccase, with a molecular mass of 66 kDa as determined by SDS-PAGE. The pH optimum for 2,2'-azino-bis-[3-ethyltiazoline-6-sulfonate] oxidation ranged between 2.5 and 3.0 in 100 mM tartrate buffer. Optimum temperature for laccase activity was determined to be around 70 °C. The kinetic of laccase was investigated with four phenolic substrates. The lowest Km values (17 and 20 µM) were found for ABTS and guaiacol, respectively. Coriolopsis gallica 1184 laccase was characterized by mass spectrometry and shows that C. gallica 1184_LacI is very likely a new member of the AA1_1 subfamily. Our results clearly show high competitive potential of the robust extracellular C. gallica 1184 laccase to use it in different industrial processes.

rpoD gene pyrosequencing for the assessment of Pseudomonas diversity in a water sample from the Woluwe River.

A water sample from a noncontaminated site at the source of the Woluwe River (Belgium) was analyzed by culture-dependent and -independent methods. Pseudomonas isolates were identified by sequencing and analysis of the rpoD gene. Cultureindependent methods consisted of cloning and pyrosequencing of a Pseudomonas rpoD amplicon from total DNA extracted from the same sample and amplified with selective rpoD gene primers. Among a total of 14,540 reads, 6,228 corresponded to Pseudomonas rpoD gene sequences by a BLAST analysis in the NCBI database. The selection criteria for the reads were sequences longer than 400 bp, an average Q40 value greater than 25, and>85% identity with a Pseudomonas species. Of the 6,228 Pseudomonas rpoD sequences, 5,345 sequences met the established criteria for selection. Sequences were clustered by phylogenetic analysis and by use of the QIIME software package. Representative sequences of each cluster were assigned by BLAST analysis to a known Pseudomonas species when the identity with the type strain was greater than or equal to 96%. Twenty-six species distributed among 12 phylogenetic groups or subgroups within the genus were detected by pyrosequencing. Pseudomonas stutzeri, P. moraviensis, and P. simiae were the only cultured species not detected by pyrosequencing. The predominant phylogenetic group within the Pseudomonas genus was the P. fluorescens group, as determined by culture-dependent and -independent analyses. In all analyses, a high number of putative novel phylospecies was found: 10 were identified in the cultured strains and 246 were detected by pyrosequencing, indicating that the diversity of Pseudomonas species has not been fully described.

Evaluation of oprI and oprL genes as molecular markers for the genus Pseudomonas and their use in studying the biodiversity of a small Belgian River.

A multiplex PCR based on oprI and oprL, coding for the outer membrane lipoprotein I and the peptidoglycan-associated lipoprotein OprL, respectively, was developed for the detection of Pseudomonas strains from a bacterial collection isolated from a small river. To study the diversity of these Pseudomonas isolates, an oprI-oprL gene sequence database of 94 Pseudomonas type strains was constructed. Phylogenetic analysis of the concatenated oprI and oprL gene sequences of the Pseudomonas type strains showed that they were largely congruent with the classification based on the MLSA approach based on 16S rRNA, gyrB, rpoB and rpoD gene sequences of Mulet et al. in 2010. Identification of the isolates demonstrated a high diversity of Pseudomonas isolates at the source of the river located in a forest of which most isolates belonged to the Pseudomonas fluorescens lineage. On the other hand, the Pseudomonas population isolated at an anthropized site at the mouth of the river, receiving waste water from both households and industry, was very different and contained many Pseudomonas aeruginosa isolates.

Formation of the conserved pseudouridine at position 55 in archaeal tRNA.

Pseudouridine (Psi) located at position 55 in tRNA is a nearly universally conserved RNA modification found in all three domains of life. This modification is catalyzed by TruB in bacteria and by Pus4 in eukaryotes, but so far the Psi55 synthase has not been identified in archaea. In this work, we report the ability of two distinct pseudouridine synthases from the hyperthermophilic archaeon Pyrococcus furiosus to specifically modify U55 in tRNA in vitro. These enzymes are (pfu)Cbf5, a protein known to play a role in RNA-guided modification of rRNA, and (pfu)PsuX, a previously uncharacterized enzyme that is not a member of the TruB/Pus4/Cbf5 family of pseudouridine synthases. (pfu)PsuX is hereafter renamed (pfu)Pus10. Both enzymes specifically modify tRNA U55 in vitro but exhibit differences in substrate recognition. In addition, we find that in a heterologous in vivo system, (pfu)Pus10 efficiently complements an Escherichia coli strain deficient in the bacterial Psi55 synthase TruB. These results indicate that it is probable that (pfu)Cbf5 or (pfu)Pus10 (or both) is responsible for the introduction of pseudouridine at U55 in tRNAs in archaea. While we cannot unequivocally assign the function from our results, both possibilities represent unexpected functions of these proteins as discussed herein.

Expression, purification, and structural prediction of the Ets transcription factor ERM.

The PEA3 group within the Ets family comprises PEA3, ER81, and ERM, three transcription factors of about 500 residues. These factors are highly conserved in their ETS DNA-binding domain and in their two transcriptional activation domains. They are involved in many developmental processes and regulate cancer development via metastasis, as in the case of some breast tumors. Here, we describe the oversynthesis of human ERM from a baculovirus expression vector in Spodoptera frugiperda (Sf9) cells, and the subsequent purification and structural characterization of this protein. Oversynthesis of ERM was confirmed by measuring band intensities on SDS-PAGE gels and by Western blot analysis. Two-step purification by affinity chromatography led to a highly stable protein. Electromobility shift assays suggested that this purified protein is functional, since it recognizes specific Ets DNA-binding sites. We then used circular dichroism and infrared spectrometry to perform a structural analysis of the purified full-length ERM, and compared the results with those of current structural prediction algorithms. Our study indicates that ERM contains a highly structured ETS-domain and suggests that each of the N- and C-terminal transactivating domains also contains an alpha-helix. In contrast, the 250-residue central domain seems to have very little structure.

Crystal structure of Bacillus subtilis TrmB, the tRNA (m7G46) methyltransferase.

The structure of Bacillus subtilis TrmB (BsTrmB), the tRNA (m7G46) methyltransferase, was determined at a resolution of 2.1 A. This is the first structure of a member of the TrmB family to be determined by X-ray crystallography. It reveals a unique variant of the Rossmann-fold methyltransferase (RFM) structure, with the N-terminal helix folded on the opposite site of the catalytic domain. The architecture of the active site and a computational docking model of BsTrmB in complex with the methyl group donor S-adenosyl-L-methionine and the tRNA substrate provide an explanation for results from mutagenesis studies of an orthologous enzyme from Escherichia coli (EcTrmB). However, unlike EcTrmB, BsTrmB is shown here to be dimeric both in the crystal and in solution. The dimer interface has a hydrophobic core and buries a potassium ion and five water molecules. The evolutionary analysis of the putative interface residues in the TrmB family suggests that homodimerization may be a specific feature of TrmBs from Bacilli, which may represent an early stage of evolution to an obligatory dimer.

Sequence-structure-function relationships of a tRNA (m7G46) methyltransferase studied by homology modeling and site-directed mutagenesis.

The Escherichia coli TrmB protein and its Saccharomyces cerevisiae ortholog Trm8p catalyze the S-adenosyl-L-methionine-dependent formation of 7-methylguanosine at position 46 (m7G46) in tRNA. To learn more about the sequence-structure-function relationships of these enzymes we carried out a thorough bioinformatics analysis of the tRNA:m7G methyltransferase (MTase) family to predict sequence regions and individual amino acid residues that may be important for the interactions between the MTase and the tRNA substrate, in particular the target guanosine 46. We used site-directed mutagenesis to construct a series of alanine substitutions and tested the activity of the mutants to elucidate the catalytic and tRNA-recognition mechanism of TrmB. The functional analysis of the mutants, together with the homology model of the TrmB structure and the results of the phylogenetic analysis, revealed the crucial residues for the formation of the substrate-binding site and the catalytic center in tRNA:m7G MTases.

Overexpression, purification, crystallization and crystallographic analysis of CopK of Cupriavidus metallidurans.

CopK of Cupriavidus metallidurans is a 93-amino-acid protein whose mature form (73 amino acids) has been purified and crystallized by the hanging-drop vapour-diffusion method in 100 mM citrate pH 3.5, 200 mM Li2SO4, 20%(w/v) glycerol, 13%(w/v) PEG 8000. Crystals display orthorhombic symmetry, with unit-cell parameters a = 57.53, b = 128.65, c = 49.77 A, and diffract to 2.2 A resolution using synchrotron radiation.

A primordial RNA modification enzyme: the case of tRNA (m1A) methyltransferase.

The modified nucleoside 1-methyladenosine (m(1)A) is found in the T-loop of many tRNAs from organisms belonging to the three domains of life (Eukaryota, Bacteria, Archaea). In the T-loop of eukaryotic and bacterial tRNAs, m(1)A is present at position 58, whereas in archaeal tRNAs it is present at position(s) 58 and/or 57, m(1)A57 being the obligatory intermediate in the biosynthesis of 1-methylinosine (m(1)I57). In yeast, the formation of m(1)A58 is catalysed by the essential tRNA (m(1)A58) methyltransferase (MTase), a tetrameric enzyme that is composed of two types of subunits (Gcd14p and Gcd10p), whereas in the bacterium Thermus thermophilus the enzyme is a homotetramer of the TrmI polypeptide. Here, we report that the TrmI enzyme from the archaeon Pyrococcus abyssi is also a homotetramer. However, unlike the bacterial site-specific TrmI MTase, the P.abyssi enzyme is region-specific and catalyses the formation of m(1)A at two adjacent positions (57 and 58) in the T-loop of certain tRNAs. The stabilisation of P.abyssi TrmI at extreme temperatures involves intersubunit disulphide bridges that reinforce the tetrameric oligomerisation, as revealed by biochemical and crystallographic evidences. The origin and evolution of m(1)A MTases is discussed in the context of different hypotheses of the tree of life.

The yggH gene of Escherichia coli encodes a tRNA (m7G46) methyltransferase.

We cloned, expressed, and purified the Escherichia coli YggH protein and show that it catalyzes the S-adenosyl-L-methionine-dependent formation of N(7)-methylguanosine at position 46 (m(7)G46) in tRNA. Additionally, we generated an E. coli strain with a disrupted yggH gene and show that the mutant strain lacks tRNA (m(7)G46) methyltransferase activity.

Yeast epiarginase regulation, an enzyme-enzyme activity control: identification of residues of ornithine carbamoyltransferase and arginase responsible for enzyme catalytic and regulatory activities.

In the presence of ornithine and arginine, ornithine carbamoyltransferase (OTCase) and arginase form a one-to-one enzyme complex in which the activity of OTCase is inhibited whereas arginase remains catalytically active. The mechanism by which these nonallosteric enzymes form a stable complex triggered by the binding of their respective substrates raises the question of how such a cooperative association is induced. Analyses of mutations in both enzymes identify residues that are required for their association, some of them being important for catalysis. In arginase, two cysteines at the C terminus of the protein are crucial for its epiarginase function but not for its catalytic activity and trimeric structure. In OTCase, mutations of putative ornithine binding residues, Asp-182, Asn-184, Asn-185, Cys-289, and Glu-256 greatly reduced the affinity for ornithine and impaired the interaction with arginase. The four lysine residues located in the SMG loop, Lys-260, Lys-263, Lys-265, and Lys-268, also play an important role in mediating the sensitivity of OTCase to ornithine and to arginase and appear to be involved in transducing and enhancing the signal given by ornithine for the closure of the catalytic domain.

Cloning and characterization of tRNA (m1A58) methyltransferase (TrmI) from Thermus thermophilus HB27, a protein required for cell growth at extreme temperatures.

N1-methyladenosine (m1A) is found at position 58 in the T-loop of many tRNAs. In yeast, the formation of this modified nucleoside is catalyzed by the essential tRNA (m1A58) methyltransferase, a tetrameric enzyme that is composed of two types of subunits (Gcd14p and Gcd10p). In this report we describe the cloning, expression and characterization of a Gcd14p homolog from the hyperthermophilic bacterium Thermus thermophilus. The purified recombinant enzyme behaves as a homotetramer of 150 kDa by gel filtration and catalyzes the site- specific formation of m1A at position 58 of the T-loop of tRNA in the absence of any other complementary protein. S-adenosylmethionine is used as donor of the methyl group. Thus, we propose to name the bacterial enzyme TrmI and accordingly its structural gene trmI. These results provide a key evolutionary link between the functionally characterized two-component eukaryotic enzyme and the recently described crystal structure of an uncharacterized, putative homotetrameric methyltransferase Rv2118c from Mycobacterium tuberculosis. Interest ingly, inactivation of the T.thermophilus trmI gene results in a thermosensitive phenotype (growth defect at 80 degrees C), which suggests a role of the N1-methylation of tRNA adenosine-58 in adaptation of life to extreme temperatures.

Preliminary X-ray crystallographic analysis of tRNA pseudouridine 55 synthase from the thermophilic eubacterium Thermotoga maritima.

Thermotoga maritima TruB, an enzyme responsible for the formation of pseudouridine in tRNA, has been purified and crystallized by the hanging-drop vapour-diffusion method in 100 mM citrate pH 3.5, 200 mM Li(2)SO(4), 20% glycerol, 13% PEG 8000. Crystals display orthorhombic symmetry, with unit-cell parameters a = 47.39, b = 83.88, c = 98.72 A, and diffract to 2.0 A resolution using synchrotron radiation. A solution was obtained by molecular replacement using part of the recently published crystal structure of Escherichia coli TruB bound to a synthetic RNA.

Overexpression, purification, crystallization and preliminary X-ray crystallographic analysis of Pseudomonas aeruginosa L-arginine deiminase.

Pseudomonas aeruginosa L-arginine deiminase, an enzyme catalyzing the irreversible catabolism of arginine to citrulline, has been produced in selenomethionyl form. The protein was purified and crystallized by the sitting-drop vapour-diffusion method using a precipitant solution consisting of 55% MPD, 100 mM cacodylate pH 6.5, 20 mM MgCl(2). Crystals display tetragonal symmetry (P4(1)2(1)2 or P4(3)2(1)2), with unit-cell parameters a = b = 106.0, c = 300.2 A, and diffract to 2.7 A resolution. A complete MAD data set was collected to 3.2 A resolution on beamline BM30 at ESRF.