Structural Characterization of Functionally Important Chloride Binding Sites in the Marine Alkaline Phosphatase.

Enzyme stability and function can be affected by various environmental factors, such as temperature, pH, and ionic strength. Enzymes that are located outside the relatively unchanging environment of the cytosol, such as those residing in the periplasmic space of bacteria or extracellularly secreted, are challenged by more fluctuations in the aqueous medium. Bacterial alkaline phosphatases (APs) are generally affected by ionic strength of the medium, but this varies substantially between species.

The high catalytic rate of the cold-active Vibrio alkaline phosphatase requires a hydrogen bonding network involving a large interface loop.

The role of surface loops in mediating communication through residue networks is still a relatively poorly understood part in the study of cold adaptation of enzymes, especially in terms of their quaternary interactions. Alkaline phosphatase (AP) from the psychrophilic marine bacterium Vibrio splendidus (VAP) is characterized by an analogous large surface loop in each monomer, referred to as the large loop, that hovers over the active site of the other monomer. It presumably has a role in the high catalytic efficiency of VAP which accompanies its extremely low thermal stability.

X-ray crystal structure of alkaline phosphatase with the non-competitive inhibitor cyclohexylamine.

Background: -nitrophenyl phosphate, the common substrate for alkaline phosphatase (AP), is available as a cyclohexylamine salt. Here, we report that cyclohexylamine is a non-competitive inhibitor of APs.

Methods: Cyclohexylamine inhibited four different APs.

Chloride promotes refolding of active alkaline phosphatase through an inactive dimeric intermediate with an altered interface.

Most enzymes are homodimers or higher order multimers. Cold-active alkaline phosphatase from (VAP) transitions into a dimer with very low activity under mild denaturation conditions. The desire to understand why this dimer fails to efficiently catalyse phosphomonoester hydrolysis led us to investigate interfacial communication between subunits.

pH-Dependent Binding of Chloride to a Marine Alkaline Phosphatase Affects the Catalysis, Active Site Stability, and Dimer Equilibrium.

The effect of ionic strength on enzyme activity and stability varies considerably between enzymes. Ionic strength is known to affect the catalytic activity of some alkaline phosphatases (APs), such as Escherichia coli AP, but how ions affect APs is debated. Here, we studied the effect of various ions on a cold-adapted AP from Vibrio splendidus (VAP).

Cold-active alkaline phosphatase is irreversibly transformed into an inactive dimer by low urea concentrations.

Alkaline phosphatase is a homodimeric metallo-hydrolase where both Zn(2+) and Mg(2+) are important for catalysis and stability. Cold-adapted alkaline phosphatase variants have high activity at low temperatures and lower thermal stability compared with variants from mesophilic hosts. The instability, and thus inactivation, could be due to loose association of the dimers and/or loosely bound Mg(2)(+) in the active site, but this has not been studied in detail for the cold-adapted variants.

Effects of different colloid infusions on ROTEM and Multiplate during elective brain tumour neurosurgery.

Background: The European Medicines Agency does not recommend the use of hydroxyethyl starch-based volume replacement solutions in critically ill patients due to an increased risk of renal failure. However, this recommendation is questionable for its perioperative use. Several recent randomised controlled studies do not indicate a risk for renal failure-not even after high-risk surgery.

Characterizing alpha helical properties of Ebola viral proteins as potential targets for inhibition of alpha-helix mediated protein-protein interactions.

Ebola, considered till recently as a rare and endemic disease, has dramatically transformed into a potentially global humanitarian crisis. The genome of Ebola, a member of the Filoviridae family, encodes seven proteins. Based on the recently implemented software (PAGAL) for analyzing the hydrophobicity and amphipathicity properties of alpha helices (AH) in proteins, we characterize the helices in the Ebola proteome.

Directed evolution induces tributyrin hydrolysis in a virulence factor of Xylella fastidiosa using a duplicated gene as a template.

Duplication of genes is one of the preferred ways for natural selection to add advantageous functionality to the genome without having to reinvent the wheel with respect to catalytic efficiency and protein stability. The duplicated secretory virulence factors of Xylella fastidiosa (LesA, LesB and LesC), implicated in Pierce's disease of grape and citrus variegated chlorosis of citrus species, epitomizes the positive selection pressures exerted on advantageous genes in such pathogens. A deeper insight into the evolution of these lipases/esterases is essential to develop resistance mechanisms in transgenic plants.

Protein structure quality assessment based on the distance profiles of consecutive backbone Cα atoms.

Predicting the three dimensional native state structure of a protein from its primary sequence is an unsolved grand challenge in molecular biology. Two main computational approaches have evolved to obtain the structure from the protein sequence - ab initio/de novo methods and template-based modeling - both of which typically generate multiple possible native state structures. Model quality assessment programs (MQAP) validate these predicted structures in order to identify the correct native state structure.

The PDB database is a rich source of alpha-helical anti-microbial peptides to combat disease causing pathogens.

The therapeutic potential of α-helical anti-microbial peptides (AH-AMP) to combat pathogens is fast gaining prominence. Based on recently published open access software for characterizing α-helical peptides (PAGAL), we elucidate a search methodology (SCALPEL) that leverages the massive structural data pre-existing in the PDB database to obtain AH-AMPs belonging to the host proteome. We provide in vitro validation of SCALPEL on plant pathogens ( Xylella fastidiosa, Xanthomonas arboricola and Liberibacter crescens) by identifying AH-AMPs that mirror the function and properties of cecropin B, a well-studied AH-AMP.

A computational module assembled from different protease family motifs identifies PI PLC from Bacillus cereus as a putative prolyl peptidase with a serine protease scaffold.

Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units.

The dipeptidyl peptidase IV inhibitors vildagliptin and K-579 inhibit a phospholipase C: a case of promiscuous scaffolds in proteins.

The long term side effects of any newly introduced drug is a subject of intense research, and often raging controversies. One such example is the dipeptidyl peptidase-IV (DPP4) inhibitor used for treating type 2 diabetes, which is inconclusively implicated in increased susceptibility to acute pancreatitis. Previously, based on a computational analysis of the spatial and electrostatic properties of active site residues, we have demonstrated that phosphoinositide-specific phospholipase C (PI-PLC) from Bacillus cereus is a prolyl peptidase using in vivo experiments.

The electrostatic profile of consecutive Cβ atoms applied to protein structure quality assessment.

The structure of a protein provides insight into its physiological interactions with other components of the cellular soup. Methods that predict putative structures from sequences typically yield multiple, closely-ranked possibilities. A critical component in the process is the model quality assessing program (MQAP), which selects the best candidate from this pool of structures.

Structural phylogeny by profile extraction and multiple superimposition using electrostatic congruence as a discriminator.

Phylogenetic analysis of proteins using multiple sequence alignment (MSA) assumes an underlying evolutionary relationship in these proteins which occasionally remains undetected due to considerable sequence divergence. Structural alignment programs have been developed to unravel such fuzzy relationships. However, none of these structure based methods have used electrostatic properties to discriminate between spatially equivalent residues.

Dynamics fingerprint and inherent asymmetric flexibility of a cold-adapted homodimeric enzyme. A case study of the Vibrio alkaline phosphatase.

Background: Protein dynamics influence protein function and stability and modulate conformational changes. Such motions depend on the underlying networks of intramolecular interactions and communicating residues within the protein structure. Here, we provide the first characterization of the dynamic fingerprint of the dimeric alkaline phosphatase (AP) from the cold-adapted Vibrio strain G15-21 (VAP), which is among the APs with the highest known kcat at low temperatures.

A measure of the broad substrate specificity of enzymes based on 'duplicate' catalytic residues.

The ability of an enzyme to select and act upon a specific class of compounds with unerring precision and efficiency is an essential feature of life. Simultaneously, these enzymes often catalyze the reaction of a range of similar substrates of the same class, and also have promiscuous activities on unrelated substrates. Previously, we have established a methodology to quantify promiscuous activities in a wide range of proteins.

Inhibition of a cold-active alkaline phosphatase by imipenem revealed by in silico modeling of metallo-β-lactamase active sites.

We demonstrate the inhibition of the native phosphatase activity of a cold active alkaline phosphatase from Vibrio (VAP) (IC(50) of 44±4 (n=4)μM at pH 7.0 after a 30min preincubation) by a specific β-lactam compound (only by imipenem, and not by ertapenem, meropenem, ampicillin or penicillin G). The homologous scaffold was detected by an in silico analysis that established the spatial and electrostatic congruence of the active site of a Class B2 CphA metallo-β-lactamase from Aeromonas hydrophila to the active site of VAP.

Cerebral blood flow and transcranial doppler sonography measurements of CO2-reactivity in acute traumatic brain injured patients.

Background: Cerebral blood flow (CBF) measurements are helpful in managing patients with traumatic brain injury (TBI), and testing the cerebrovascular reactivity to CO(2) provides information about injury severity and outcome. The complexity and potential hazard of performing CBF measurements limits routine clinical use. An alternative approach is to measure the CBF velocity using bedside, non-invasive, and transcranial Doppler (TCD) sonography.

Isolation and biochemical characterisation of lipid rafts from Atlantic cod (Gadus morhua) intestinal enterocytes.

Lipid rafts are glycosphingolipid/cholesterol-enriched membrane microdomains that have been extensively studied during the past two decades. Our aim was to isolate and perform biochemical characterization of lipid rafts from the intestinal brush border membrane (BBM) of Atlantic cod (Gadus morhua) to confirm their existence in a cold-water species and compare their characteristics with lipid rafts from other species in terms of lipid and protein content. To validate the isolation process, we assayed marker enzymes for subcellular organelles, including alkaline phosphatase (AP) and leucine aminopeptidase (LAP), both well-known marker enzymes for BBM and lipid rafts.

The 1.4 A crystal structure of the large and cold-active Vibrio sp. alkaline phosphatase.

Alkaline phosphatase (AP) from the cold-adapted Vibrio strain G15-21 is among the AP variants with the highest known k(cat) value. Here the structure of the enzyme at 1.4 A resolution is reported and compared to APs from E.

Effects of replacing active site residues in a cold-active alkaline phosphatase with those found in its mesophilic counterpart from Escherichia coli.

Alkaline phosphatase (AP) from a North Atlantic marine Vibrio bacterium was previously characterized as being kinetically cold-adapted. It is still unknown whether its characteristics originate locally in the active site or are linked to more general structural factors. There are three metal-binding sites in the active site of APs, and all three metal ions participate in catalysis.

Engineered disulfide bonds increase active-site local stability and reduce catalytic activity of a cold-adapted alkaline phosphatase.

Alkaline phosphatase is an extracellular enzyme that is membrane-bound in eukaryotes but resides in the periplasmic space of bacteria. It normally carries four cysteine residues that form two disulfide bonds, for instance in the APs of Escherichia coli and vertebrates. An AP variant from a Vibrio sp.

Microscopic rate-constants for substrate binding and acylation in cold-adaptation of trypsin I from Atlantic cod.

Temperature imposes limits on where life can thrive and this is evident in the evolution of the basic structural properties of proteins. Cold-adaptation of enzymes is one example, where the catalytic rate constant (k(cat)) is increased compared with hot-acclimated homologous under identical assay conditions. Trypsin I from Atlantic cod (Gadus morhua) has catalytic efficiency (k(cat)/K(m)) for amide hydrolysis that is 17-fold larger than observed for bovine trypsin.