Membrane Targeted Azobenzene Drives Optical Modulation of Bacterial Membrane Potential.

Recent studies have shown that bacterial membrane potential is dynamic and plays signaling roles. Yet, little is still known about the mechanisms of membrane potential dynamics regulation-owing to a scarcity of appropriate research tools. Optical modulation of bacterial membrane potential could fill this gap and provide a new approach for studying and controlling bacterial physiology and electrical signaling. Here, the authors show that a membrane-targeted azobenzene (Ziapin2) can be used to photo-modulate the membrane potential in cells of the Gram-positive bacterium Bacillus subtilis. It is found that upon exposure to blue-green light (λ = 470 nm), isomerization of Ziapin2 in the bacteria membrane induces hyperpolarization of the potential. To investigate the origin of this phenomenon, ion-channel-deletion strains and ion channel blockers are examined. The authors found that in presence of the chloride channel blocker idanyloxyacetic acid-94 (IAA-94) or in absence of KtrAB potassium transporter, the hyperpolarization response is attenuated. These results reveal that the Ziapin2 isomerization can induce ion channel opening in the bacterial membrane and suggest that Ziapin2 can be used for studying and controlling bacterial electrical signaling. This new optical tool could contribute to better understand various microbial phenomena, such as biofilm electric signaling and antimicrobial resistance.

Rational backbone redesign of a fructosyl peptide oxidase to widen its active site access tunnel.

Fructosyl peptide oxidases (FPOXs) are enzymes currently used in enzymatic assays to measure the concentration of glycated hemoglobin and albumin in blood samples, which serve as biomarkers of diabetes. However, since FPOX are unable to work directly on glycated proteins, current enzymatic assays are based on a preliminary proteolytic digestion of the target proteins. Herein, to improve the speed and costs of the enzymatic assays for diabetes testing, we applied a rational design approach to engineer a novel enzyme with a wider access tunnel to the catalytic site, using a combination of Rosetta design and molecular dynamics simulations. Our final design, L3_35A, shows a significantly wider and shorter access tunnel, resulting from the deletion of five-amino acids lining the gate structures and from a total of 35 point mutations relative to the wild-type (WT) enzyme. Indeed, upon experimental testing, our engineered enzyme shows good structural stability and maintains significant activity relative to the WT.

Integration of bio-responsive silver in 1D photonic crystals: towards the colorimetric detection of bacteria.

The colour purity and versatility of fabrication of one-dimensional photonic crystals (1D PhCs) make them ideal candidates for colorimetric sensing of a variety of analytes. For instance, the detection of bacterial contaminants in food via colorimetric sensors can be highly appealing, as most of the existing detection techniques are in general time-consuming and the read-out requires specialised personnel. Here, we present a colorimetric sensor based on hybrid plasmonic/photonic 1D crystals. We demonstrate that the modification of the silver plasmon resonance brought about by the effective silver/bacterium interaction can be translated into the visible spectral region, producing a change in the structural colour. In addition, we observe a superior colorimetric sensitivity against the Gram negative Escherichia coli compared to the Gram positive Micrococcus luteus, a result that we attribute to the more efficient electrostatic interaction and cellular adhesion between the silver surface and the Gram-negative bacteria outer membrane. This approach demonstrates that in principle an easy colorimetric detection of bacterial contaminants can be achieved through the use of bio-responsive plasmonic materials, such as silver, whose selective electrostatic interaction with bacterial cell wall is well-known and occurs without the need of chemical functionalisation.

Structural characterization of human O-phosphoethanolamine phospho-lyase.

Human O-phosphoethanolamine phospho-lyase (hEtnppl; EC 4.2.3.2) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the degradation of O-phosphoethanolamine (PEA) into acetaldehyde, phosphate and ammonia. Physiologically, the enzyme is involved in phospholipid metabolism, as PEA is the precursor of phosphatidylethanolamine in the CDP-ethanolamine (Kennedy) pathway. Here, the crystal structure of hEtnppl in complex with pyridoxamine 5'-phosphate was determined at 2.05 Šresolution by molecular replacement using the structure of A1RDF1 from Arthrobacter aurescens TC1 (PDB entry 5g4i) as the search model. Structural analysis reveals that the two proteins share the same general fold and a similar arrangement of active-site residues. These results provide novel and useful information for the complete characterization of the human enzyme.

Insight into GEBR-32a: Chiral Resolution, Absolute Configuration and Enantiopreference in PDE4D Inhibition.

Alzheimer's disease is the most common type of dementia, affecting millions of people worldwide. One of its main consequences is memory loss, which is related to downstream effectors of cyclic adenosine monophosphate (cAMP). A well-established strategy to avoid cAMP degradation is the inhibition of phosphodiesterase (PDE). In recent years, GEBR-32a has been shown to possess selective inhibitory properties against PDE type 4 family members, resulting in an improvement in spatial memory processes without the typical side effects that are usually correlated with this mechanism of action. In this work, we performed the HPLC chiral resolution and absolute configuration assignment of GEBR-32a. We developed an efficient analytical and semipreparative chromatographic method exploiting an amylose-based stationary phase, we studied the chiroptical properties of both enantiomers and we assigned their absolute configuration by 1H-NMR (nuclear magnetic resonance). Lastly, we measured the IC50 values of both enantiomers against both the PDE4D catalytic domain and the long PDE4D3 isoform. Results strongly support the notion that GEBR-32a inhibits the PDE4D enzyme by interacting with both the catalytic pocket and the regulatory domains.

Structure-Based Virtual Screening Allows the Identification of Efficient Modulators of E-Cadherin-Mediated Cell-Cell Adhesion.

Cadherins are a large family of transmembrane calcium-dependent cell adhesion proteins that orchestrate adherens junction formation and are crucially involved in tissue morphogenesis. Due to their important role in cancer development and metastasis, cadherins can be considered attractive targets for drug discovery. A recent crystal structure of the complex of a cadherin extracellular portion and a small molecule inhibitor allowed the identification of a druggable interface, thus providing a viable strategy for the design of cadherin dimerization modulators. Here, we report on a structure-based virtual screening approach that led to the identification of efficient and selective modulators of E-cadherin-mediated cell-cell adhesion. Of all the putative inhibitors that were identified and experimentally tested by cell adhesion assays using human pancreatic tumor BxPC-3 cells expressing both E-cadherin and P-cadherin, two compounds turned out to be effective in inhibiting stable cell-cell adhesion at micromolar concentrations. Moreover, at the same concentrations, one of them also showed anti-invasive properties in cell invasion assays. These results will allow further development of novel and selective cadherin-mediated cell-cell adhesion modulators for the treatment of a variety of cadherin-expressing solid tumors and for improving the efficiency of drug delivery across biological barriers.

Biohybrid Electrospun Membrane for the Filtration of Ketoprofen Drug from Water.

A current challenge in materials science and biotechnology is to express a specific and controlled functionality on the large interfacial area of a nanostructured material to create smart biohybrid systems for targeted applications. Here, we report on a biohybrid system featuring poly(vinyl alcohol) as the supporting synthetic polymer and bovine serum albumin as the biofunctional element. The optimal processing conditions to produce these self-standing composite membranes are determined, and the composition and distribution of the bioactive agent within the polymeric matrices are analyzed. A post-processing cross-linking using glutaraldehyde enables this functional membrane to be used as a chemical filter in aqueous environments. By demonstrating that our mats can remove large amounts of ketoprofen from water, we show that the combination of a BSA-induced biofunctionality with a nanostructured fibrous material allows for the development of an efficient biohybrid filtering device for the large and widely used family of nonsteroidal anti-inflammatory drugs (NSAIDs). The crystal structure of the complex between BSA and ketoprofen is determined for the first time and confirms the interaction between the two species.

Hybrid One-Dimensional Plasmonic-Photonic Crystals for Optical Detection of Bacterial Contaminants.

Photonic crystal-based biosensors hold great promise as low-cost devices for real-time monitoring of a variety of biotargets, for example, bacterial contaminants in food. Here, we report the proof-of-concept for a new colorimetric sensor of bacterial contamination, which is based on a novel hybrid plasmonic-photonic device. Our system consists of a layer of silver, a plasmonic metal exhibiting a well-known bioactivity, on top of a one-dimensional photonic crystal. We attribute the bioresponsivity to the formation of polarization charges at the Ag/bacterium interface within a sort of "bio-doping" mechanism. Interestingly, this triggers a blue shift in the photonic response. As an example, we assessed the validity of our approach by detecting one of the most hazardous contaminants, Escherichia coli. This work demonstrates that our device can be a low-cost and portable platform for the detection of common bacterial contaminants.

Synthesis and Structure-Activity relationship of 1-(5-isoquinolinesulfonyl)piperazine analogues as inhibitors of Mycobacterium tuberculosis IMPDH.

Tuberculosis (TB) is a major infectious disease associated increasingly with drug resistance. Thus, new anti-tubercular agents with novel mechanisms of action are urgently required for the treatment of drug-resistant TB. In prior work, we identified compound 1 (cyclohexyl(4-(isoquinolin-5-ylsulfonyl)piperazin-1-yl)methanone) and showed that its anti-tubercular activity is attributable to inhibition of inosine-5'-monophosphate dehydrogenase (IMPDH) in Mycobacterium tuberculosis. In the present study, we explored the structure-activity relationship around compound 1 by synthesizing and evaluating the inhibitory activity of analogues against M. tuberculosis IMPDH in biochemical and whole-cell assays. X-ray crystallography was performed to elucidate the mode of binding of selected analogues to IMPDH. We establish the importance of the cyclohexyl, piperazine and isoquinoline rings for activity, and report the identification of an analogue with IMPDH-selective activity against a mutant of M. tuberculosis that is highly resistant to compound 1. We also show that the nitrogen in urea analogues is required for anti-tubercular activity and identify benzylurea derivatives as promising inhibitors that warrant further investigation.

Molecular Bases of PDE4D Inhibition by Memory-Enhancing GEBR Library Compounds.

Selected members of the large rolipram-related GEBR family of type 4 phosphodiesterase (PDE4) inhibitors have been shown to facilitate long-term potentiation and to improve memory functions without causing emetic-like behavior in rodents. Despite their micromolar-range binding affinities and their promising pharmacological and toxicological profiles, few if any structure-activity relationship studies have been performed to elucidate the molecular bases of their action. Here, we report the crystal structure of a number of GEBR library compounds in complex with the catalytic domain of PDE4D as well as their inhibitory profiles for both the long PDE4D3 isoform and the catalytic domain alone. Furthermore, we assessed the stability of the observed ligand conformations in the context of the intact enzyme using molecular dynamics simulations. The longer and more flexible ligands appear to be capable of forming contacts with the regulatory portion of the enzyme, thus possibly allowing some degree of selectivity between the different PDE4 isoforms.

Thermal stabilization of the deglycating enzyme Amadoriase I by rational design.

Amadoriases are a class of FAD-dependent enzymes that are found in fungi, yeast and bacteria and that are able to hydrolyze glycated amino acids, cleaving the sugar moiety from the amino acidic portion. So far, engineered Amadoriases have mostly found practical application in the measurement of the concentration of glycated albumin in blood samples. However, these engineered forms of Amadoriases show relatively low absolute activity and stability levels, which affect their conditions of use. Therefore, enzyme stabilization is desirable prior to function-altering molecular engineering. In this work, we describe a rational design strategy based on a computational screening method to evaluate a library of potentially stabilizing disulfide bonds. Our approach allowed the identification of two thermostable Amadoriase I mutants (SS03 and SS17) featuring a significantly higher T50 (55.3 °C and 60.6 °C, respectively) compared to the wild-type enzyme (52.4 °C). Moreover, SS17 shows clear hyperstabilization, with residual activity up to 95 °C, whereas the wild-type enzyme is fully inactive at 55 °C. Our computational screening method can therefore be considered as a promising approach to expedite the design of thermostable enzymes.

Review: Engineering of thermostable enzymes for industrial applications.

The catalytic properties of some selected enzymes have long been exploited to carry out efficient and cost-effective bioconversions in a multitude of research and industrial sectors, such as food, health, cosmetics, agriculture, chemistry, energy, and others. Nonetheless, for several applications, naturally occurring enzymes are not considered to be viable options owing to their limited stability in the required working conditions. Over the years, the quest for novel enzymes with actual potential for biotechnological applications has involved various complementary approaches such as mining enzyme variants from organisms living in extreme conditions (extremophiles), mimicking evolution in the laboratory to develop more stable enzyme variants, and more recently, using rational, computer-assisted enzyme engineering strategies. In this review, we provide an overview of the most relevant enzymes that are used for industrial applications and we discuss the strategies that are adopted to enhance enzyme stability and/or activity, along with some of the most relevant achievements. In all living species, many different enzymes catalyze fundamental chemical reactions with high substrate specificity and rate enhancements. Besides specificity, enzymes also possess many other favorable properties, such as, for instance, cost-effectiveness, good stability under mild pH and temperature conditions, generally low toxicity levels, and ease of termination of activity. As efficient natural biocatalysts, enzymes provide great opportunities to carry out important chemical reactions in several research and industrial settings, ranging from food to pharmaceutical, cosmetic, agricultural, and other crucial economic sectors.

Structural investigations on orotate phosphoribosyltransferase from Mycobacterium tuberculosis, a key enzyme of the de novo pyrimidine biosynthesis.

The Mycobacterium tuberculosis orotate phosphoribosyltransferase (MtOPRT) catalyses the conversion of α-D-5-phosphoribosyl-1-pyrophosphate (PRPP) and orotate (OA) in pyrophosphate and orotidine 5'-monophosphate (OMP), in presence of Mg2+. This enzyme is the only responsible for the synthesis of orotidine 5'-monophosphate, a key precursor in the de novo pyrimidine biosynthesis pathway, making MtOPRT an attractive drug target for the development of antitubercular agents. We report the crystal structures of MtOPRT in complex with PRPP (2.25 Å resolution), inorganic phosphate (1.90 Å resolution) and the exogenous compound Fe(III) dicitrate (2.40 Å resolution). The overall structure of the mycobacterial enzyme is highly similar to those described for other OPRTases, with the "flexible loop" assuming a well define conformation and making specific contacts with the Fe(III)-dicitrate complex. The structures here reported add to the knowledge of a potential drug target for tuberculosis, and will provide a useful tool for the structure-based drug design of potent enzyme inhibitors.

Biochemical and structural investigations on phosphoribosylpyrophosphate synthetase from Mycobacterium smegmatis.

Mycobacterium smegmatis represents one model for studying the biology of its pathogenic relative Mycobacterium tuberculosis. The structural characterization of a M. tuberculosis ortholog protein can serve as a valid tool for the development of molecules active against the M. tuberculosis target. In this context, we report the biochemical and structural characterization of M. smegmatis phosphoribosylpyrophosphate synthetase (PrsA), the ortholog of M. tuberculosis PrsA, the unique enzyme responsible for the synthesis of phosphoribosylpyrophosphate (PRPP). PRPP is a key metabolite involved in several biosynthetic pathways including those for histidine, tryptophan, nucleotides and decaprenylphosphoryl-arabinose, an essential precursor for the mycobacterial cell wall biosynthesis. Since M. tuberculosis PrsA has been validated as a drug target for the development of antitubercular agents, the data presented here will add to the knowledge of the mycobacterial enzyme and could contribute to the development of M. tuberculosis PrsA inhibitors of potential pharmacological interest.

Essential but Not Vulnerable: Indazole Sulfonamides Targeting Inosine Monophosphate Dehydrogenase as Potential Leads against Mycobacterium tuberculosis.

A potent, noncytotoxic indazole sulfonamide was identified by high-throughput screening of >100,000 synthetic compounds for activity against Mycobacterium tuberculosis (Mtb). This noncytotoxic compound did not directly inhibit cell wall biogenesis but triggered a slow lysis of Mtb cells as measured by release of intracellular green fluorescent protein (GFP). Isolation of resistant mutants followed by whole-genome sequencing showed an unusual gene amplification of a 40 gene region spanning from Rv3371 to Rv3411c and in one case a potential promoter mutation upstream of guaB2 (Rv3411c) encoding inosine monophosphate dehydrogenase (IMPDH). Subsequent biochemical validation confirmed direct inhibition of IMPDH by an uncompetitive mode of inhibition, and growth inhibition could be rescued by supplementation with guanine, a bypass mechanism for the IMPDH pathway. Beads containing immobilized indazole sulfonamides specifically interacted with IMPDH in cell lysates. X-ray crystallography of the IMPDH-IMP-inhibitor complex revealed that the primary interactions of these compounds with IMPDH were direct pi-pi interactions with the IMP substrate. Advanced lead compounds in this series with acceptable pharmacokinetic properties failed to show efficacy in acute or chronic murine models of tuberculosis (TB). Time-kill experiments in vitro suggest that sustained exposure to drug concentrations above the minimum inhibitory concentration (MIC) for 24 h were required for a cidal effect, levels that have been difficult to achieve in vivo. Direct measurement of guanine levels in resected lung tissue from tuberculosis-infected animals and patients revealed 0.5-2 mM concentrations in caseum and normal lung tissue. The high lesional levels of guanine and the slow lytic, growth-rate-dependent effect of IMPDH inhibition pose challenges to developing drugs against this target for use in treating TB.

The Inosine Monophosphate Dehydrogenase, GuaB2, Is a Vulnerable New Bactericidal Drug Target for Tuberculosis.

VCC234718, a molecule with growth inhibitory activity against Mycobacterium tuberculosis (Mtb), was identified by phenotypic screening of a 15344-compound library. Sequencing of a VCC234718-resistant mutant identified a Y487C substitution in the inosine monophosphate dehydrogenase, GuaB2, which was subsequently validated to be the primary molecular target of VCC234718 in Mtb. VCC234718 inhibits Mtb GuaB2 with a Ki of 100 nM and is uncompetitive with respect to IMP and NAD+. This compound binds at the NAD+ site, after IMP has bound, and makes direct interactions with IMP; therefore, the inhibitor is by definition uncompetitive. VCC234718 forms strong pi interactions with the Y487 residue side chain from the adjacent protomer in the tetramer, explaining the resistance-conferring mutation. In addition to sensitizing Mtb to VCC234718, depletion of GuaB2 was bactericidal in Mtb in vitro and in macrophages. When supplied at a high concentration (≥125 µM), guanine alleviated the toxicity of VCC234718 treatment or GuaB2 depletion via purine salvage. However, transcriptional silencing of guaB2 prevented Mtb from establishing an infection in mice, confirming that Mtb has limited access to guanine in this animal model. Together, these data provide compelling validation of GuaB2 as a new tuberculosis drug target.

Crystal structure of human aldehyde dehydrogenase 1A3 complexed with NAD+ and retinoic acid.

The aldehyde dehydrogenase family 1 member A3 (ALDH1A3) catalyzes the oxidation of retinal to the pleiotropic factor retinoic acid using NAD+. The level of ALDHs enzymatic activity has been used as a cancer stem cell marker and seems to correlate with tumour aggressiveness. Elevated ALDH1A3 expression in mesenchymal glioma stem cells highlights the potential of this isozyme as a prognosis marker and drug target. Here we report the first crystal structure of human ALDH1A3 complexed with NAD+ and the product all-trans retinoic acid (REA). The tetrameric ALDH1A3 folds into a three domain-based architecture highly conserved along the ALDHs family. The structural analysis revealed two different and coupled conformations for NAD+ and REA that we propose to represent two snapshots along the catalytic cycle. Indeed, the isoprenic moiety of REA points either toward the active site cysteine, or moves away adopting the product release conformation. Although ALDH1A3 shares high sequence identity with other members of the ALDH1A family, our structural analysis revealed few peculiar residues in the 1A3 isozyme active site. Our data provide information into the ALDH1As catalytic process and can be used for the structure-based design of selective inhibitors of potential medical interest.

An active site-tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase.

Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that `close' the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Šresolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an `open' structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site-tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS.

Strict reaction and substrate specificity of AGXT2L1, the human O-phosphoethanolamine phospho-lyase.

Dysregulated expression of the AGXT2L1 gene has been associated to neuropsychiatric disorders. Recently the gene product was shown to possess O-phosphoethanolamine phospho-lyase activity. We here analyze the specificity of AGXT2L1 in terms of both reaction and substrate. We show that the enzyme, despite having evolved from a transaminase ancestor, is at least 500-fold more active as a lyase than as an aminotransferase. Furthermore, the lyase reaction is very selective for O-phosphoethanolamine, strongly discriminating against closely related compounds, and we dissect the factors that contribute to such narrow substrate specificity. Overall, AGXT2L1 function appears to be rigidly confined to phospholipid metabolism, which is altered in neuropsychiatric disturbances.

Recombinant production of eight human cytosolic aminotransferases and assessment of their potential involvement in glyoxylate metabolism.

PH1 (primary hyperoxaluria type 1) is a severe inborn disorder of glyoxylate metabolism caused by a functional deficiency of the peroxisomal enzyme AGXT (alanine-glyoxylate aminotransferase), which converts glyoxylate into glycine using L-alanine as the amino-group donor. Even though pre-genomic studies indicate that other human transaminases can convert glyoxylate into glycine, in PH1 patients these enzymes are apparently unable to compensate for the lack of AGXT, perhaps due to their limited levels of expression, their localization in an inappropriate cell compartment or the scarcity of the required amino-group donor. In the present paper, we describe the cloning of eight human cytosolic aminotransferases, their recombinant expression as His6-tagged proteins and a comparative study on their ability to transaminate glyoxylate, using any standard amino acid as an amino-group donor. To selectively quantify the glycine formed, we have developed and validated an assay based on bacterial GO (glycine oxidase); this assay allows the detection of enzymes that produce glycine by transamination in the presence of mixtures of potential amino-group donors and without separation of the product from the substrates. We show that among the eight enzymes tested, only GPT (alanine transaminase) and PSAT1 (phosphoserine aminotransferase 1) can transaminate glyoxylate with good efficiency, using L-glutamate (and, for GPT, also L-alanine) as the best amino-group donor. These findings confirm that glyoxylate transamination can occur in the cytosol, in direct competition with the conversion of glyoxylate into oxalate. The potential implications for the treatment of primary hyperoxaluria are discussed.