Surface Engineering of Escherichia coli to Display Its Phytase (AppA) and Functional Analysis of Enzyme Activities.

Escherichia coli phytase (AppA) is widely used as an exogenous enzyme in monogastric animal feed mainly because of its ability to degrade phytic acid or its salt (phytate), a natural source of phosphorus. Currently, successful recombinant production of soluble AppA has been achieved by gene overexpression using both bacterial and yeast systems. However, some methods for the biomembrane immobilization of phytases (including AppA), such as surface display on yeast cells and bacterial spores, have been investigated to avoid expensive enzyme purification processes. This study explored a homologous protein production approach for displaying AppA on the cell surface of E. coli by engineering its outer membrane (OM) for extracellular expression. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of total bacterial lysates and immunofluorescence microscopy of non-permeabilized cells revealed protein expression, whereas activity assays using whole cells or OM fractions indicated functional enzyme display, as evidenced by consistent hydrolytic rates on typical substrates (i.e., p-nitrophenyl phosphate and phytic acid). Furthermore, the in vitro results obtained using a simple method to simulate the gastrointestinal tract of poultry suggest that the whole-cell biocatalyst has potential as a feed additive. Overall, our findings support the notion that biomembrane-immobilized enzymes are reliable for the hydrolysis of poorly digestible substrates relevant to animal nutrition.

Structural Insights into the Giardia lamblia Target of Rapamycin Homolog: A Bioinformatics Approach.

TOR proteins, also known as targets of rapamycin, are serine/threonine kinases involved in various signaling pathways that regulate cell growth. The protozoan parasite Giardia lamblia is the causative agent of giardiasis, a neglected infectious disease in humans. In this study, we used a bioinformatics approach to examine the structural features of GTOR, a G. lamblia TOR-like protein, and predict functional associations. Our findings confirmed that it shares significant similarities with functional TOR kinases, including a binding domain for the FKBP-rapamycin complex and a kinase domain resembling that of phosphatidylinositol 3-kinase-related kinases. In addition, it can form multiprotein complexes such as TORC1 and TORC2. These results provide valuable insights into the structure-function relationship of GTOR, highlighting its potential as a molecular target for controlling G. lamblia cell proliferation. Furthermore, our study represents a step toward rational drug design for specific anti-giardiasis therapeutic agents.

In Silico Structural Analysis of Serine Carboxypeptidase Nf314, a Potential Drug Target in Naegleria fowleri Infections.

Naegleria fowleri, also known as the "brain-eating" amoeba, is a free-living protozoan that resides in freshwater bodies. This pathogenic amoeba infects humans as a casual event when swimming in contaminated water. Upon inhalation, N. fowleri invades the central nervous system and causes primary amoebic meningoencephalitis (PAM), a rapidly progressive and often fatal disease. Although PAM is considered rare, reducing its case fatality rate compels the search for pathogen-specific proteins with a structure-function relationship that favors their application as targets for discovering new or improved drugs against N. fowleri infections. Herein, we report a computational approach to study the structural features of Nf314 (a serine carboxypeptidase that is a virulence-related protein in N. fowleri infections) and assess its potential as a drug target, using bioinformatics tools and in silico molecular docking experiments. Our findings suggest that Nf314 has a ligand binding site suitable for the structure-based design of specific inhibitors. This study represents a further step toward postulating a reliable therapeutic target to treat PAM with drugs specifically aimed at blocking the pathogen proliferation by inhibiting protein function.

Molecular Analysis of Streptomycin Resistance Genes in Clinical Strains of Mycobacterium tuberculosis and Biocomputational Analysis of the MtGidB L101F Variant.

Globally, tuberculosis (TB) remains a prevalent threat to public health. In 2019, TB affected 10 million people and caused 1.4 million deaths. The major challenge for controlling this infectious disease is the emergence and spread of drug-resistant Mycobacterium tuberculosis, the causative agent of TB. The antibiotic streptomycin is not a current first-line anti-TB drug. However, WHO recommends its use in patients infected with a streptomycin-sensitive strain. Several mutations in the M. tuberculosisrpsL, rrs and gidB genes have proved association with streptomycin resistance. In this study, we performed a molecular analysis of these genes in clinical isolates to determine the prevalence of known or novel mutations. Here, we describe the genetic analysis outcome. Furthermore, a biocomputational analysis of the MtGidB L101F variant, the product of a novel mutation detected in gidB during molecular analysis, is also reported as a theoretical approach to study the apparent genotype-phenotype association.

Bioinformatic Analysis of Two TOR (Target of Rapamycin)-Like Proteins Encoded by Entamoeba histolytica Revealed Structural Similarities with Functional Homologs.

The target of rapamycin (TOR), also known as FKBP-rapamycin associated protein (FRAP), is a protein kinase belonging to the PIKK (phosphatidylinositol 3-kinase (PI3K)-related kinases) family. TOR kinases are involved in several signaling pathways that control cell growth and proliferation. Entamoeba histolytica, the protozoan parasite that causes human amoebiasis, contains two genes encoding TOR-like proteins: EhFRAP and EhTOR2. To assess their potential as drug targets to control the cell proliferation of E. histolytica, we studied the structural features of EhFRAP and EhTOR2 using a biocomputational approach. The overall results confirmed that both TOR amoebic homologs share structural similarities with functional TOR kinases, and show inherent abilities to form TORC complexes and participate in protein-protein interaction networks. To our knowledge, this study represents the first in silico characterization of the structure-function relationships of EhFRAP and EhTOR2.

Structure-Function Relationship Study of a Secretory Amoebic Phosphatase: A Computational-Experimental Approach.

Phosphatases are hydrolytic enzymes that cleave the phosphoester bond of numerous substrates containing phosphorylated residues. The typical classification divides them into acid or alkaline depending on the pH at which they have optimal activity. The histidine phosphatase (HP) superfamily is a large group of functionally diverse enzymes characterized by having an active-site His residue that becomes phosphorylated during catalysis. HP enzymes are relevant biomolecules due to their current and potential application in medicine and biotechnology. Entamoeba histolytica, the causative agent of human amoebiasis, contains a gene (EHI_146950) that encodes a putative secretory acid phosphatase (EhHAPp49), exhibiting sequence similarity to histidine acid phosphatase (HAP)/phytase enzymes, i.e., branch-2 of HP superfamily. To assess whether it has the potential as a biocatalyst in removing phosphate groups from natural substrates, we studied the EhHAPp49 structural and functional features using a computational-experimental approach. Although the combined outcome of computational analyses confirmed its structural similarity with HP branch-2 proteins, the experimental results showed that the recombinant enzyme (rEhHAPp49) has negligible HAP/phytase activity. Nonetheless, results from supplementary activity evaluations revealed that rEhHAPp49 exhibits Mg2+-dependent alkaline pyrophosphatase activity. To our knowledge, this study represents the first computational-experimental characterization of EhHAPp49, which offers further insights into the structure-function relationship and the basis for future research.

Functional Display of an Amoebic Chitinase in Escherichia coli Expressing the Catalytic Domain of EhCHT1 on the Bacterial Cell Surface.

Poor solubility is the main drawback of the direct industrial exploitation of chitin, the second most abundant biopolymer after cellulose. Chemical methods are conventional to solubilize chitin from natural sources. Enzymatic hydrolysis of soluble chitinous substrates is a promising approach to obtain value-added by-products, such as N-acetylglucosamine units or low molecular weight chito-oligomers. Protein display on the bacterial membrane remains attractive to produce active enzymes anchored to a biological surface. The Lpp-OmpA system, a gene fusion of the Lpp signal sequence with the OmpA transmembrane region, represents the traditional system for targeting enzymes to the E. coli surface. EhCHT1, the amoebic chitinase, exhibits an efficient endochitinolytic activity and significant biochemical features, such as stability over a wide range of pH values. Using an extended Lpp-OmpA system as a protein carrier, we engineered E. coli to express the catalytic domain of EhCHT1 on the surface and assess the endochitinase activity as a trait. Engineered bacteria showed a consistent hydrolytic rate over a typical substrate, suggesting that the displayed enzyme has operational stability. This study supports the potential of biomembrane-associated biocatalysts as a reliable technology for the hydrolysis of soluble chitinous substrates.

A molecular dynamics approach on the Y393C variant of protein disulfide isomerase A1.

Human protein disulfide isomerase A1 (PDIA1) shows both catalytic (i.e., oxidoreductase) and non-catalytic (i.e., chaperone) activities and plays a crucial role in the oxidative folding of proteins within the endoplasmic reticulum. PDIA1 dysregulation is a common trait in numerous pathophysiological conditions, including neurodegenerative disorders and cancerous diseases. The 1178A>G mutation of the human PDIA1-encoding gene is a non-synonymous single nucleotide polymorphism detected in patients with Cole-Carpenter syndrome type 1 (CSS1), a particularly rare bone disease. In vitro studies showed that the encoded variant (PDIA1 Y393C) exhibits limited oxidoreductase activity. To gain knowledge on the structure-function relationship, we undertook a molecular dynamics (MD) approach to examine the structural stability of PDIA1 Y393C. Results showed that significant conformational changes are the structural consequence of the amino acid substitution Tyr>Cys at position 393 of the PDIA1 protein. This structure-based study provides further knowledge about the molecular origin of CCS1.