Biophysical Characterization of a Novel Phosphopentomutase from the Hyperthermophilic Archaeon .

Phosphopentomutases catalyze the isomerization of ribose 1-phosphate and ribose 5-phosphate. , a hyperthermophilic archaeon, harbors a novel enzyme (PPM) that exhibits high homology with phosphohexomutases but has no significant phosphohexomutase activity. Instead, PPM catalyzes the interconversion of ribose 1-phosphate and ribose 5-phosphate.

RrA, an enzyme from Rhodospirillum rubrum, is a prototype of a new family of short-chain L-asparaginases.

L-Asparaginases (ASNases) catalyze the hydrolysis of L-Asn to L-Asp and ammonia. Members of the ASNase family are used as drugs in the treatment of leukemia, as well as in the food industry. The protomers of bacterial ASNases typically contain 300-400 amino acids (typical class 1 ASNases).

Mechanism of selective recognition of Lys48-linked polyubiquitin by macrocyclic peptide inhibitors of proteasomal degradation.

Post-translational modification of proteins with polyubiquitin chains is a critical cellular signaling mechanism in eukaryotes with implications in various cellular states and processes. Unregulated ubiquitin-mediated protein degradation can be detrimental to cellular homeostasis, causing numerous diseases including cancers. Recently, macrocyclic peptides were developed that selectively target long Lysine-48-linked polyubiquitin chains (tetra-ubiquitin) to inhibit ubiquitin-proteasome system, leading to attenuation of tumor growth in vivo.

The E. coli L-asparaginase V27T mutant: structural and functional characterization and comparison with theoretical predictions.

Bacterial L-asparaginases have been used for over 40 years as anticancer drugs. Ardalan et al. (Medical Hypotheses 112, 7-17, 2018) proposed that the V27T mutant of Escherichia coli type II L-asparaginase, EcAII(V27T), should display altered biophysical and catalytic properties compared to the wild-type enzyme, EcAII(wt), rendering it more favourable as a pharmaceutical.

The dimeric form of bacterial l-asparaginase YpAI is fully active.

l-asparaginases from mesophilic bacteria (ASNases), including two enzymes very successfully used in the treatment of leukaemia, have been consistently described as homotetramers. On the contrary, structural studies show that homodimers of these enzymes should be sufficient to carry out the catalytic reaction. In this report, we investigated whether the type I Yersinia pestis asparaginase (YpAI) is active in a dimeric form or whether the tetrameric quaternary structure is critical for its activity.

Structural and biochemical properties of L-asparaginase.

l-Asparaginase (a hydrolase converting l-asparagine to l-aspartic acid) was the first enzyme to be used in clinical practice as an anticancer agent after its approval in 1978 as a component of a treatment protocol for childhood acute lymphoblastic leukemia. Structural and biochemical properties of l-asparaginases have been extensively investigated during the last half-century, providing an accurate structural description of the enzyme isolated from a variety of sources, as well as clarifying the mechanism of its activity. This review provides a critical assessment of the current state of knowledge of primarily structural, but also selected biochemical properties of 'bacterial-type' l-asparaginases from different organisms.

Generalized enzymatic mechanism of catalysis by tetrameric L-asparaginases from mesophilic bacteria.

The mechanism of catalysis by the L-glutaminase-asparaginase from Pseudomonas 7A (PGA) was investigated using structural, mass spectrometry, and kinetic data. We had previously proposed mechanism of hydrolysis of L-Asn by the type II L-asparaginase from E. coli (EcAII), but that work was limited to just one enzyme.

Mechanism of Catalysis by l-Asparaginase.

Two bacterial type II l-asparaginases, from and , have played a critical role for more than 40 years as therapeutic agents against juvenile leukemias and lymphomas. Despite a long history of successful pharmacological applications and the apparent simplicity of the catalytic reaction, controversies still exist regarding major steps of the mechanism. In this report, we provide a detailed description of the reaction catalyzed by type II l-asparaginase (EcAII).

Geometric considerations support the double-displacement catalytic mechanism of l-asparaginase.

Twenty crystal structures of the complexes of l-asparaginase with l-Asn, l-Asp, and succinic acid that are currently available in the Protein Data Bank, as well as 11 additional structures determined in the course of this project, were analyzed in order to establish the level of conservation of the geometric parameters describing interactions between the substrates and the active site of the enzymes. We found that such stereochemical relationships are highly conserved, regardless of the organism from which the enzyme was isolated, specific crystallization conditions, or the nature of the ligands. Analysis of the geometry of the interactions, including Bürgi-Dunitz and Flippin-Lodge angles, indicated that Thr12 (Escherichia coli asparaginase II numbering) is optimally placed to be the primary nucleophile in the most likely scenario utilizing a double-displacement mechanism, whereas catalysis through a single-displacement mechanism appears to be the least likely.

Opportunistic complexes of E. coli L-asparaginases with citrate anions.

Active sites of enzymes are highly optimized for interactions with specific substrates, thus binding of opportunistic ligands is usually observed only in the absence of native substrates or products. However, during growth of crystals required for structure determination enzymes are often exposed to conditions significantly divergent from the native ones, leading to binding of unexpected ligands to active sites even in the presence of substrates. Failing to recognize this possibility may lead to incorrect interpretation of experimental results and to faulty conclusions.

Crystal Structure of the Labile Complex of IL-24 with the Extracellular Domains of IL-22R1 and IL-20R2.

Crystal structure of the ternary complex of human IL-24 with two receptors, IL-22R1 and IL-20R2, has been determined at 2.15 Å resolution. A crystallizable complex was created by a novel approach involving fusing the ligand with a flexible linker to the presumed low-affinity receptor, and coexpression of this construct in S2 cells together with the presumed high-affinity receptor.

Human β-defensin 4 - defensin without the "twist".

β-defensins are small, cysteine-rich, cationic peptides that contribute to various processes related to both arms of host defense, the innate and adaptive immunities. All β-defensins are potent antimicrobials with activity targeting a broad range of pathogens. Some human β-defensins (hBDs) are also capable of binding and activating specific chemokine receptors, leading to chemotaxis of receptor-presenting cells.

Structural analysis and unique molecular recognition properties of a Bauhinia forficata lectin that inhibits cancer cell growth.

Unlabelled: Lectins have been used at length for basic research and clinical applications. New insights into the molecular recognition properties enhance our basic understanding of carbohydrate-protein interactions and aid in the design/development of new lectins. In this study, we used a combination of cell-based assays, glycan microarrays, and X-ray crystallography to evaluate the structure and function of the recombinant Bauhinia forficata lectin (BfL).

Crystal Structure of a Complex of the Intracellular Domain of Interferon λ Receptor 1 (IFNLR1) and the FERM/SH2 Domains of Human JAK1.

The crystal structure of a construct consisting of the FERM and SH2-like domains of the human Janus kinase 1 (JAK1) bound to a fragment of the intracellular domain of the interferon-λ receptor 1 (IFNLR1) has been determined at the nominal resolution of 2.1Å. In this structure, the receptor peptide forms an 85-Å-long extended chain, in which both the previously identified box1 and box2 regions bind simultaneously to the FERM and SH2-like domains of JAK1.

Structure of a lectin from the sea mussel Crenomytilus grayanus (CGL).

CGL is a 150 amino-acid residue lectin that was originally isolated from the sea mussel Crenomytilus grayanus. It is specific for binding GalNAc/Gal-containing carbohydrate moieties and in general does not share sequence homology with other known galectins or lectins. Since CGL displays antibacterial, antifungal and antiviral activities, and interacts with high affinity with mucin-type receptors, which are abundant on some cancer cells, knowledge of its structure is of significant interest.

Design of composite inhibitors targeting glutamate carboxypeptidase II: the importance of effector functionalities.

Unlabelled: Inhibitors targeting human glutamate carboxypeptidase II (GCPII) typically consist of a P1' glutamate-derived binding module, which warrants the high affinity and specificity, linked to an effector function that is positioned within the entrance funnel of the enzyme. Here we present a comprehensive structural and computational study aimed at dissecting the importance of the effector function for GCPII binding and affinity. To this end we determined crystal structures of human GCPII in complex with a series of phosphoramidate-based inhibitors harboring effector functions of diverse physicochemical characteristics.

Structural and biochemical characterization of a novel aminopeptidase from human intestine.

N-acetylated α-linked acidic dipeptidase-like protein (NAALADase L), encoded by the NAALADL1 gene, is a close homolog of glutamate carboxypeptidase II, a metallopeptidase that has been intensively studied as a target for imaging and therapy of solid malignancies and neuropathologies. However, neither the physiological functions nor structural features of NAALADase L are known at present. Here, we report a thorough characterization of the protein product of the human NAALADL1 gene, including heterologous overexpression and purification, structural and biochemical characterization, and analysis of its expression profile.

Structural and biochemical characterization of the folyl-poly-γ-l-glutamate hydrolyzing activity of human glutamate carboxypeptidase II.

In addition to its well-characterized role in the central nervous system, human glutamate carboxypeptidase II (GCPII; Uniprot ID Q04609) acts as a folate hydrolase in the small intestine, participating in the absorption of dietary polyglutamylated folates (folyl-n-γ-l-glutamic acid), which are the provitamin form of folic acid (also known as vitamin B9 ). Despite the role of GCPII as a folate hydrolase, nothing is known about the processing of polyglutamylated folates by GCPII at the structural or enzymological level. Moreover, many epidemiologic studies on the relationship of the naturally occurring His475Tyr polymorphism to folic acid status suggest that this polymorphism may be associated with several pathologies linked to impaired folate metabolism.

Structural characterization of P1'-diversified urea-based inhibitors of glutamate carboxypeptidase II.

Urea-based inhibitors of human glutamate carboxypeptidase II (GCPII) have advanced into clinical trials for imaging metastatic prostate cancer. In parallel efforts, agents with increased lipophilicity have been designed and evaluated for targeting GCPII residing within the neuraxis. Here we report the structural and computational characterization of six complexes between GCPII and P1'-diversified urea-based inhibitors that have the C-terminal glutamate replaced by more hydrophobic moieties.

Preclinical evaluation of human secretoglobin 3A2 in mouse models of lung development and fibrosis.

Secretoglobin (SCGB) 3A2 is a member of the SCGB gene superfamily of small secreted proteins, predominantly expressed in lung airways. We hypothesize that human SCGB3A2 may exhibit anti-inflammatory, growth factor, and antifibrotic activities and be of clinical utility. Recombinant human SCGB3A2 was expressed, purified, and biochemically characterized as a first step to its development as a therapeutic agent in clinical settings.

Human β-defensin-3 structure motifs that are important in CXCR4 antagonism.

Previously, we reported that human β-defensin (hBD)-3 can both antagonize CXCR4 function on T cells and promote receptor internalization in the absence of activation. In the present study, we explored the important structural elements of hBD-3 that are involved in blocking CXCR4 activation by its natural ligand, stromal-derived factor 1α (SDF-1α; CXCL12). Results from site-directed mutagenesis studies suggest that the ability of hBD-3 to inhibit SDF-1α-CXCR4 interaction, as assayed either by blocking SDF-1 binding to CXCR4 or antagonizing SDF-1-induced Ca(2+) mobilization, is correlated with the presence of hBD-3 cysteine residues, specific surface-distributed cationic residues, and the electrostatic properties and availability of both hBD-3 termini.

Sometimes it takes two to tango: contributions of dimerization to functions of human α-defensin HNP1 peptide.

Human myeloid α-defensins called HNPs play multiple roles in innate host defense. The Trp-26 residue of HNP1 was previously shown to contribute importantly to its ability to kill S. aureus, inhibit anthrax lethal factor (LF), bind gp120 of HIV-1, dimerize, and undergo further self-association.

Novel substrate-based inhibitors of human glutamate carboxypeptidase II with enhanced lipophilicity.

Virtually all low molecular weight inhibitors of human glutamate carboxypeptidase II (GCPII) are highly polar compounds that have limited use in settings where more lipophilic molecules are desired. Here we report the identification and characterization of GCPII inhibitors with enhanced liphophilicity that are derived from a series of newly identified dipeptidic GCPII substrates featuring nonpolar aliphatic side chains at the C-terminus. To analyze the interactions governing the substrate recognition by GCPII, we determined crystal structures of the inactive GCPII(E424A) mutant in complex with selected dipeptides and complemented the structural data with quantum mechanics/molecular mechanics calculations.

Novel peptides based on HIV-1 gp120 sequence with homology to chemokines inhibit HIV infection in cell culture.

The sequential interaction of the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) with CD4 and certain chemokine coreceptors initiates host cell entry of the virus. The appropriate chemokines have been shown to inhibit viral replication by blocking interaction of the gp120 envelope protein with the coreceptors. We considered the possibility that this interaction involves a motif of the gp120 that may be structurally homologous to the chemokines.