Single-gene disorders: what role could moonlighting enzymes play?

Single-gene disorders with "simple" Mendelian inheritance do not always imply that there will be an easy prediction of the phenotype from the genotype, which has been shown for a number of metabolic disorders. We propose that moonlighting enzymes (i.e., metabolic enzymes with additional functional activities) could contribute to the complexity of such disorders. The lack of knowledge about the additional functional activities of proteins could result in a lack of correlation between genotype and phenotype. In this review, we highlight some notable and recent examples of moonlighting enzymes and their possible contributions to human disease. Because knowledge and cataloging of the moonlighting activities of proteins are essential for the study of cellular function and human physiology, we also review recently reported and recommended methods for the discovery of moonlighting activities.     

Active site-independent recognition of substrates and product by bovine prothrombinase: a fluorescence resonance energy transfer study

The conversion of prothrombin to thrombin is catalyzed by prothrombinase, an enzyme complex composed of the serine proteinase factor Xa and a cofactor protein, factor Va, assembled on membranes. Kinetic studies indicate that interactions with extended macromolecular recognition sites (exosites) rather than the active site of prothrombinase are the principal determinants of binding affinity for substrate or product. We now provide a model-independent evaluation of such ideas by physical studies of the interaction of substrate derivatives and product with prothrombinase. The enzyme complex was assembled using Xa modified with a fluorescent peptidyl chloromethyl ketone to irreversibly occlude the active site. Binding was inferred by prethrombin 2-dependent perturbations in the fluorescence of Oregon Green(488) at the active site of prothrombinase. Active site-independent binding was also unequivocally established by fluorescence resonance energy transfer between 2,6-dansyl tethered to the active site of Xa and eosin tethered to the active sites of either thrombin or meizothrombin des fragment 1. Comparable interprobe distances obtained from these measurements suggest that substrate and product interact equivalently with the enzyme. Competition established the ability of a range of substrate or product derivatives to bind in a mutually exclusive fashion to prothrombinase. Equilibrium dissociation constants obtained for the active site-independent binding of prothrombin, prethrombin 2, meizothrombin des fragment 1 and thrombin to prothrombinase were comparable with their affinities inferred from kinetic studies using active enzyme. Our findings directly establish that binding affinity is principally determined by the exosite-mediated interaction of either the substrate, both possible intermediates, or product with prothrombinase. A single type of exosite binding interaction evidently drives affinity and binding specificity through the stepwise reactions necessary for the two cleavage reactions of prothrombin activation and product release.     

Substitutions at the Asp-473 latch residue of chlamydomonas ribulosebisphosphate carboxylase/oxygenase cause decreases in carboxylation efficiency and CO(2)/O(2) specificity

The loop between alpha-helix 6 and beta-strand 6 in the alpha/beta-barrel active site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) plays a key role in discriminating between gaseous substrates CO(2) and O(2). Based on numerous x-ray crystal structures, loop 6 is either closed or open depending on the presence or absence, respectively, of substrate ligands. The carboxyl terminus folds over loop 6 in the closed conformation, prompting speculation that it may trigger or latch loop 6 closure. Because an x-ray crystal structure of tobacco Rubisco revealed that phosphate is located at a site in the open form that is occupied by the carboxyl group of Asp-473 in the closed form, it was proposed that Asp-473 may serve as the latch that holds the carboxyl terminus over loop 6. To assess the essentiality of Asp-473 in catalysis, we used directed mutagenesis and chloroplast transformation of the green alga Chlamydomonas reinhardtii to create D473A and D473E mutant enzymes. The D473A and D473E mutant strains can grow photoautotrophically, indicating that Asp-473 is not essential for catalysis. However, both substitutions caused 87% decreases in carboxylation catalytic efficiency (V(max)/K(m)) and approximately 16% decreases in CO(2)/O(2) specificity. If the carboxyl terminus is required for stabilizing loop 6 in the closed conformation, there must be additional residues at the carboxyl terminus/loop 6 interface that contribute to this mechanism. Considering that substitutions at residue 473 can influence CO(2)/O(2) specificity, further study of interactions between loop 6 and the carboxyl terminus may provide clues for engineering an improved Rubisco.     

Structure and transport mechanism of the bacterial oxalate transporter OxlT

Membrane proteins that belong to the major facilitator superfamily (MFS) are found in organisms across the evolutionary spectrum and mediate the transport of a variety of substrates ranging from small metabolites to neurotransmitters. The oxalate transporter (OxlT) is a representative MFS protein, and exchanges formate for oxalate across the cytoplasmic membrane of the organism Oxalobacter formigenes. Here, we present a structural model for the protein conformational changes that occur during oxalate transport by combining a three-dimensional map of the oxalate-bound, "closed" state of OxlT at 6.5 A determined by cryo-electron microscopy with a model of the "open" state of OxlT based on the atomic structures of the related transporters, glycerol-3-phosphate transporter (GlpT) and lactose permease (LacY). We demonstrate that the principal structural change associated with substrate transport is a concerted rocking movement of the two structurally similar halves of the protein relative to each other. Our structural model places two positively charged residues, Arg-272 and Lys-355 in the central cavity, suggesting that electrostatic interactions between these residues and the oxalate anion is a key step in generating the conformational change between the open and closed states of the transporter.     

Three-dimensional electron microscopic imaging of membrane invaginations in Escherichia coli overproducing the chemotaxis receptor Tsr

Electron tomography is a powerful method for determining the three-dimensional structures of large macromolecular assemblies, such as cells, organelles, and multiprotein complexes, when crystallographic averaging methods are not applicable. Here we used electron tomographic imaging to determine the molecular architecture of Escherichia coli cells engineered to overproduce the bacterial chemotaxis receptor Tsr. Tomograms constructed from fixed, cryosectioned cells revealed that overproduction of Tsr led to formation of an extended internal membrane network composed of stacks and extended tubular structures. We present an interpretation of the tomogram in terms of the packing arrangement of Tsr using constraints derived from previous X-ray and electron-crystallographic studies of receptor clusters. Our results imply that the interaction between the cytoplasmic ends of Tsr is likely to stabilize the presence of the membrane networks in cells overproducing Tsr. We propose that membrane invaginations that are potentially capable of supporting axial interactions between receptor clusters in apposing membranes could also be present in wild-type E. coli and that such receptor aggregates could play an important role in signal transduction during bacterial chemotaxis.     

In vitro antibacterial activity in seed extracts of Manilkara zapota, Anona squamosa, and Tamarindus indica

Extracts prepared from seeds of Manilkara zapota, Anona squamosa, and Tamarindus indica were screened for their antibacterial activity by disc diffusion and broth dilution methods. Acetone and methanol extracts of T. indica seeds were found active against both gram-positive and gram-negative organisms. MIC values of potent extracts against susceptible organisms ranged from 53-380 μg/mL. Methanol extract of T. indica and acetone extract of M. zapota seeds were found to be bactericidal.     

CD44 differentially activates mouse NK T cells and conventional T cells

NK T (NKT) cells are an important component of the innate immune system and recognize the MHC class I-like CD1d molecule. NKT cells possess significant immunoregulatory activity due to their rapid secretion of large quantities of pro- and anti-inflammatory cytokines following CD1d-dependent stimulation. Because the innate immune system is programmed to respond to a multitude of diverse stimuli and must be able to quickly differentiate between pathogenic and endogenous signals, we hypothesized that, apart from stimulation via the TCR (e.g., CD1d-dependent activation), there must be multiple activation pathways that can be triggered through other cell surface receptors on NKT cells. Therefore, we analyzed the ability of CD44, a structurally diverse cell surface receptor expressed on most cells, to stimulate murine NKT cells, compared with conventional T cells. Stimulation of CD44 through Ab cross-linking or binding to its natural ligands hyaluronan and osteopontin induced NKT cells to secrete cytokines, up-regulate activation markers, undergo morphological changes, and resist activation-induced cell death, whereas conventional T cells only exhibited changes in morphology and protection from activation-induced cell death. This CD44-specific stimulation of NKT cells correlated with their ability to bind hyaluronan. Thus, fundamental differences in CD44 function between these lymphocyte subsets suggest an important biological role for CD44 in the innate immune response.     

Lipoxin A4 inhibits immune cell binding to salivary epithelium and vascular endothelium

Lipoxins are formed by leukocytes during cell-cell interactions with epithelial or endothelial cells. Native lipoxin A(4) (LXA(4)) binds to the G protein-coupled lipoxin receptors formyl peptide receptor 2 (FPR2)/ALX and CysLT1. Furthermore, LXA(4) inhibits recruitment of neutrophils, by attenuating chemotaxis, adhesion, and transmigration across vascular endothelial cells. LXA(4) thus appears to serve as an endogenous "stop signal" for immune cell-mediated tissue injury (Serhan CN; Annu Rev Immunol 25: 101-137, 2007). The role of LXA(4) has not been addressed in salivary epithelium, and little is known about its effects on vascular endothelium. Here, we determined that interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) receptor activation in vascular endothelium and salivary epithelium upregulated the expression of adhesion molecules that facilitates the binding of immune cells. We hypothesize that the activation of the ALX/FPR2 and/or CysLT1 receptors by LXA(4) decreases this cytokine-mediated upregulation of cell adhesion molecules that enhance lymphocyte binding to both the vascular endothelium and salivary epithelium. In agreement with this hypothesis, we observed that nanomolar concentrations of LXA(4) blocked IL-1β- and TNF-α-mediated upregulation of E-selectin and intercellular cell adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVECs). Binding of Jurkat cells to stimulated HUVECs was abrogated by LXA(4). Furthermore, LXA(4) preincubation with human submandibular gland cell line (HSG) also blocked TNF-α-mediated upregulation of vascular cell adhesion molecule-1 (VCAM-1) in these cells, and it reduced lymphocyte adhesion. These findings suggest that ALX/FPR2 and/or CysLT1 receptor activation in endothelial and epithelial cells blocks cytokine-induced adhesion molecule expression and consequent binding of lymphocytes, a critical event in the pathogenesis of Sj?gren's syndrome (SS).     

Commentary: The carboxyl-terminal Crk SH3 domain: Regulatory strategies and new perspectives

Since their discovery as cellular counterparts of viral oncogenes more than two decades ago, enormous progress has been made in unraveling the complex regulatory pathways of signal transduction initiated by the Crk family of proteins. New structural and biochemical studies have uncovered novel insights into both negative and positive regulation of Crk mediated by its atypical carboxyl-terminal SH3 domain (SH3C). Moreover, SH3C is tyrosine phosphorylated by receptor tyrosine kinases and non-receptor tyrosine kinases, thereby permitting assemblages of other SH2/PTB domain containing proteins. Such non-canonical signaling by the Crk SH3C reveals new regulatory strategies for adaptor proteins.     

Molecular hybridization of bioactives: Synthesis and antitubercular evaluation of novel dibenzofuran embodied homoisoflavonoids via Baylis–Hillman reaction

A novel series of natural product like dibenzofuran embodied homoisoflavonoids [(E)-3-(dibenzo[b,d]furan-2-ylmethylene)chroman-4-ones] designed by molecular hybridization were synthesized in very good yields via a sequence of reactions involving base catalyzed Baylis–Hillmann (BH) reaction of 2-dibenzofuran carboxaldehyde and methyl acrylate; bromination of BH adduct; condensation of resulted allylic bromide with substituted phenols or 2-dibenzofuranol followed by cyclization. Among the all 11 new compounds screened for in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv (MTB), (E)-3-(dibenzo[b,d]furan-2-ylmethylene)-6-fluorochroman-4-one (7f) and (E)-3-(dibenzo[b,d] furan-2-ylmethylene)-6-fluorochroman-4-one (7g) were found to be active with MIC 12.5 μg/mL.