The structure of a calcium-dependent phosphoinositide-specific phospholipase C fromPseudomonassp. 62186, the first from a Gram-negative bacterium

Abstract

Bacterial phosphoinositide-specific phospholipases C (PI-PLCs) are the smallest members of the PI-PLC family, which includes much larger mammalian enzymes responsible for signal transduction as well as enzymes from protozoan parasites, yeast and plants. Eukaryotic PI-PLCs have calcium in the active site, but this is absent in the known structures of Gram-positive bacteria, where its role is instead played by arginine. In addition to their use in a number of industrial applications, the bacterial enzymes attract special interest because they can serve as convenient models of the catalytic domains of eukaryotic enzymes for in vitro activity studies. Here, the structure of a PI-PLC from Pseudomonas sp. 62186 is reported, the first from a Gram-negative bacterium and the first of a native bacterial PI-PLC with calcium present in the active site. Solution of the structure posed particular problems owing to the low sequence identity of available homologous structures. Its dependence on calcium for catalysis makes this enzyme a better model for studies of the mammalian PI-PLCs than the previously used calcium-independent bacterial PI-PLCs.

Search and Subvert: Minimalist Bacterial Phosphatidylinositol-Specific Phospholipase C Enzymes

“…54 In contrast, the Gram-negative bacterial PI-PLC have acidic side chains that can chelate Ca 2+ , and these active sites are very similar to those found in mammalian PI-PLCs. 9 PI-PLC structures from the Gram-positive extracellular pathogens Bacillus sp. (PDB 1gym, 1t6m) and S. aureus obtained at pH 7.5 (PDB 3v18) show the same general architecture and excellent structural similarity with most differences concentrated in the surface loops.…”

“…and the Gram-positive bacterial PI-PLC structures are much more striking. 9 The imperfect barrel is preserved in Str. antibioticus PI-PLC (PDB 3h4x), but it has eight helices unlike the Gram-positive structures which have only six.…”

“…8 In contrast, PI-PLCs secreted by Grampositive and Gram-negative bacteria are minimalist enzymes encapsulating membrane binding and enzyme activity in the (βα) 8 barrel itself. 6,9 The first of these minimalist PI-PLCs, as well as nonspecific PLC and sphingomyelinase activities, was identified in the culture supernatants of the Gram-positive bacteria Bacillus cereus in 1965. 10 Such fractionated bacterial extracts with PI-cleaving activities were early tools in exploring the asymmetric distribution of phospholipids in eukaryotic cell plasma membranes.…”

Structural Dynamics and Catalytic Properties of a Multimodular Xanthanase

“…Cloning, Expression, and Purification. The PspXan9 gene was inserted into a Bacillus expression plasmid essentially as described for a phospholipase 36 with an affinity tag sequence to ease the purification process. Briefly, the DNA encoding the mature polypeptide predicted by SignalP 37 was cloned in frame to the Bacillus clausii secretion signal, BcSP, replacing the native secretion signal sequence followed by a polyhistidine tag (HHHHHHPR).…”

Structure and Dynamics of a Promiscuous Xanthan Lyase from Paenibacillus nanensis and the Design of Variants with Increased Stability and Activity

“…Cloning and Expression of PXL The gene encoding PXL (GenBank: MG561391) was amplified by PCR from genomic DNA of the bacterium Paenibacillus nanensis isolated from a forest soil sample from China. Partial sequencing of the 16S rRNA gene of this Paenibacillus nanensis, revealed 98.1% identity to the type strain Paenibacillus nanensis (GenBank: AB265206) The xanthan lyase gene was inserted into a Bacillus expression plasmid as described in (Moroz et al, 2017) with an affinity tag sequence to simplify the purification process. Briefly, the DNA encoding the mature polypeptide predicted by Signal P (Bendtsen et al, 2004) was cloned (with In-Fusion HD EcoDry Cloning Kit) in frame to the Bacillus clausii secretion signal, BcSP, replacing the native secretion signal sequence followed by a polyhistidine tag (HHHHHHPR).…”