Crystal structure of Pseudomonas aeruginosa lipase in the open conformation - The prototype for family I.1 of bacterial lipases

The x-ray structure of the lipase from Pseudomonas aeruginosa PAO1 has been determined at 2.54 Angstrom resolution. It is the first structure of a member of homology family I.1 of bacterial lipases. The structure shows a variant of the alpha/beta hydrolase fold, with Ser(82), Asp(229), and His(251) as the catalytic triad residues. Compared with the "canonical" alpha/beta hydrolase fold, the first two P-strands and one alpha-helix (alpha E) are not present. The absence of helix alpha E allows the formation of a stabilizing intramolecular disulfide bridge. The loop containing His251 is stabilized by an octahedrally coordinated calcium ion. On top of the active site a lid subdomain is in an open conformation, making the catalytic cleft accessible from the solvent region. A triacylglycerol analogue is covalently bound to Ser(82) in the active site, demonstrating the position of the oxyanion hole and of the three pockets that accommodate the sn-1, sn-2, and sn-3 fatty acid chains. The inhibited enzyme can be thought to mimic the structure of the tetrahedral intermediate that occurs during the acylation step of the reaction. Analysis of the binding mode of the inhibitor suggests that the size of the acyl pocket and the size and interactions of the sn-2 binding pocket are the predominant determinants of the regio- and enantio-preference of the enzyme

Arts and Humanities: Senate Report (1976): Report 02

<p>(A) Translation profile of wild-type M5bG7 (top diagram, original profile) and upon introduction of translational attenuation sites (bottom diagram, adapted profile) in <i>E</i>. <i>coli</i>. Secondary structure alignment (β-sheets—blue bars, α-helices—dark red bars, uncolored empty space—linking structural elements) of M5bG7 to EH-Ar to identify regions for introduction of slow-translating stretches (indicated with arrows). The domains in M5bG7 were delineated based on the domain architecture of EH-Ar represented in the color code as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127039#pone.0127039.g001" target="_blank">Fig 1A</a>. Note the longer N-terminal domain of M5bG7 than that of EH-Ar. (B) Representative immunoblot of M5bG7 (abbreviated M5) variants. L1 (Leu 189, CUC/A, numbering is according to the M5bG7 sequence) and L2 (Leu 257, CUG/A) denote the synonymous exchange of a fast-translating Leu codons to Leu CUA in the first and second attenuation, respectively, and LL (Leu189, CUC/A, Leu257, CUG/A) in both simultaneously. T, total protein, S, soluble and I, insoluble fraction. GAPDH served as loading control. (C) Quantification of immunoblots of three biological replicates ± SEM. **, p<0.01, Tukey’s test. For details refer to the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127039#pone.0127039.g001" target="_blank">Fig 1</a>. (D) Quantification of mRNA levels by qRT-PCR of the M5bG7 variants. Values were normalized to GAPDH mRNA expression, represented as a fold-change to the wild-type mRNA and are means ± SEM (n = 3).</p

Modifying the stereochemistry of an enzyme-catalyzed reaction by directed evolution

Aldolases have potential as tools for the synthesis of stereochemically complex carbohydrates. Here, we show that directed evolution can be used to alter the stereochemical course of the reaction catalyzed by tagatose-1,6-bisphosphate aldolase. After three rounds of DNA shuffling and screening, the evolved aldolase showed an 80-fold improvement in k-cat/K-m toward the non-natural substrate fructose 1,6-bisphosphate, resulting in a 100-fold change in stereospecificity. (31)P NMR spectroscopy was used to show that, in the synthetic direction, the evolved aldolase catalyzes the formation of carbon—carbon bonds with unnatural diastereoselectivity, where the >99:<1 preference for the formation of tagatose 1,6-bisphosphate was switched to a 4:1 preference for the diastereoisomer, fructose 1,6-bisphosphate. This demonstration is of considerable significance to synthetic chemists requiring efficient syntheses of complex stereoisomeric products, such as carbohydrate mimetics

Optimization of translation profiles enhances protein expression and solubility.

mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs) and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5'-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein

Crystal Structure of Pseudomonas aeruginosa Lipase in the Open Conformation. The Prototype for Family I.1 of Bacterial Lipases

The x-ray structure of the lipase from Pseudomonas aeruginosa PAO1 has been determined at 2.54 Å resolution. It is the first structure of a member of homology family I.1 of bacterial lipases. The structure shows a variant of the α/β hydrolase fold, with Ser82, Asp229, and His251 as the catalytic triad residues. Compared with the “canonical” α/β hydrolase fold, the first two β-strands and one α-helix (αE) are not present. The absence of helix αE allows the formation of a stabilizing intramolecular disulfide bridge. The loop containing His251 is stabilized by an octahedrally coordinated calcium ion. On top of the active site a lid subdomain is in an open conformation, making the catalytic cleft accessible from the solvent region. A triacylglycerol analogue is covalently bound to Ser82 in the active site, demonstrating the position of the oxyanion hole and of the three pockets that accommodate the sn-1, sn-2, and sn-3 fatty acid chains. The inhibited enzyme can be thought to mimic the structure of the tetrahedral intermediate that occurs during the acylation step of the reaction. Analysis of the binding mode of the inhibitor suggests that the size of the acyl pocket and the size and interactions of the sn-2 binding pocket are the predominant determinants of the regio- and enantio-preference of the enzyme.

Translation attenuation sites delineate domain boundaries and impact protein solubility.

<p>(A) Translation profile of EH-Ar predicted with RiboTempo. Vertical gray bars represent the rate of translation of each single codon which is averaged (red line) along the whole ORF with a window of 19 codons [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127039#pone.0127039.ref042" target="_blank">42</a>]. Translation minima below the genome-wide threshold (blue horizontal line) denote the putative slow-translating attenuation sites. AA denotes amino acid number, kDa marks the corresponding molecular weight and SS denotes the predicted secondary structure (β-sheets—blue bars, α-helices—dark red bars, uncolored empty space—linking structural elements). The rainbow-colored bar visualizes the putative structural domains, colored in the same way in the 3D- structure (PDB 1EHY). (B) Summary of the exchanged codons in EH-Ar. The position of the exchanged amino acids for each variant is indicated. (C) Translation profiles of EH-Ar variants with exchanged slow translating patches (B) predicted with RiboTempo. (D, E). Removal of the translational attenuation sites reduces the solubility of EH-Ar. (D) Representative immunoblot of EH-Ar variants (summarized in C). The total (T) protein content was fractionated into soluble (S) and insoluble (I) fractions and 0.05 OD₆₀₀ of cells were applied per lane. GAPDH served as a loading control; note that it is a completely soluble protein and its absence in the insoluble fraction confirms the good quality of the fractionation procedure. (E) Quantification of the immunoblots of three biological replicates ± SEM. Each total fraction was normalized to GAPDH intensity to allow for comparison between the samples; the soluble and insoluble fractions were determined as a percentage of this normalized value. *, p<0.05, Tukey’s test. (F) Quantification of mRNA levels by qRT-PCR of the EH-Ar variants. Values were normalized to GAPDH mRNA expression, represented as a fold-change to the wild-type mRNA and are means ± SEM (n = 3).</p