25 research outputs found
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
Isolation and in silico characterization of novel esterase gene with β-lactamase fold isolated from metagenome of north western Himalayas
Selection of Enantioselective Acyl Transfer Catalysts from a Pooled Peptide Library through a Fluorescence-Based Activity Assay: An Approach to Kinetic Resolution of Secondary Alcohols of Broad Structural Scope
Crystal Structure of Proteus mirabilis Lipase, a Novel Lipase from the Proteus/Psychrophilic Subfamily of Lipase Family I.1
Enhanced Protein Production in <i>Escherichia coli </i>by Optimization of Cloning Scars at the Vector-Coding Sequence Junction
Protein
production in Escherichia coli is a
fundamental activity for a large fraction of academic, pharmaceutical,
and industrial research laboratories. Maximum production is usually
sought, as this reduces costs and facilitates downstream purification
steps. Frustratingly, many coding sequences are poorly expressed even
when they are codon-optimized and expressed from vectors with powerful
genetic elements. In this study, we show that poor expression can
be caused by certain nucleotide sequences (e.g., cloning scars) at
the junction between the vector and the coding sequence. Since these
sequences lie between the Shine–Dalgarno sequence and the start
codon, they are an integral part of the translation initiation region.
To identify the most optimal sequences, we devised a simple and inexpensive
PCR-based step that generates sequence variants at the vector–coding
sequence junction. These sequence variants modulated expression by
up to 1000-fold. FACS-seq analyses indicated that low GC content and
relaxed mRNA stability (Δ<i>G</i>) in this region
were important, but not the only, determinants for high expression