Spectrochemistry of Firefly Bioluminescence.

The chemical reactions underlying the emission of light in fireflies and other bioluminescent beetles are some of the most thoroughly studied processes by scientists worldwide. Despite these remarkable efforts, fierce academic arguments continue around even some of the most fundamental aspects of the reaction mechanism behind the beetle bioluminescence. In an attempt to reach a consensus, we made an exhaustive search of the available literature and compiled the key discoveries on the fluorescence and chemiluminescence spectrochemistry of the emitting molecule, the firefly oxyluciferin, and its chemical analogues reported over the past 50+ years. The factors that affect the light emission, including intermolecular interactions, solvent polarity, and electronic effects, were analyzed in the context of both the reaction mechanism and the different colors of light emitted by different luciferases. The collective data points toward a combined emission of multiple coexistent forms of oxyluciferin as the most probable explanation for the variation in color of the emitted light. We also highlight realistic research directions to eventually address some of the remaining questions related to firefly bioluminescence. It is our hope that this extensive compilation of data and detailed analysis will not only consolidate the existing body of knowledge on this important phenomenon but will also aid in reaching a wider consensus on some of the mechanistic details of firefly bioluminescence.

Optogenetic control of the lac operon for bacterial chemical and protein production.

Control of the lac operon with isopropyl ß-D-1-thiogalactopyranoside (IPTG) has been used to regulate gene expression in Escherichia coli for countless applications, including metabolic engineering and recombinant protein production. However, optogenetics offers unique capabilities, such as easy tunability, reversibility, dynamic induction strength and spatial control, that are difficult to obtain with chemical inducers. We have developed a series of circuits for optogenetic regulation of the lac operon, which we call OptoLAC, to control gene expression from various IPTG-inducible promoters using only blue light. Applying them to metabolic engineering improves mevalonate and isobutanol production by 24% and 27% respectively, compared to IPTG induction, in light-controlled fermentations scalable to at least two-litre bioreactors. Furthermore, OptoLAC circuits enable control of recombinant protein production, reaching yields comparable to IPTG induction but with easier tunability of expression. OptoLAC circuits are potentially useful to confer light control over other cell functions originally designed to be IPTG-inducible.

Data Analysis of Infection Rates Among Exposed Healthcare Workers Could Reveal Effective Prophylactics Against SARS-CoV-2.

The global crisis provoked by the SARS-CoV-2 pandemic, and the economic and social consequences associated to the essential policies applied to contain it, necessitates the expedited development of therapeutic solutions. It is a priority to produce data both rapidly and accurately in order to identify current therapies that can be repurposed to offer protection from SARS-CoV-2 infection. As healthcare workers are both at high risk for infection and able to be readily diagnosed, they offer a potential wealth of data to be analyzed. A systematic data analysis of exposure and infection rates among healthcare workers could yield patterns identifying common protective factors, such as medications with prophylactic potential against SARS-CoV-2, that can be fast-tracked into available therapies. With results suggesting their activity against other coronaviruses and their widespread adoption, Antiretroviral cocktails could be a promising initial target for such large-scale data analysis approach.

Disulfide Engineered Lipase to Enhance the Catalytic Activity: A Structure-Based Approach on BTL2.

Enhancement, control, and tuning of hydrolytic activity and specificity of lipases are major goals for the industry. Thermoalkaliphilic lipases from the I.5 family, with their native advantages such as high thermostability and tolerance to alkaline pHs, are a target for biotechnological applications. Although several strategies have been applied to increase lipases activity, the enhancement through protein engineering without compromising other capabilities is still elusive. Lipases from the I.5 family suffer a unique and delicate double lid restructuration to transition from a closed and inactive state to their open and enzymatically active conformation. In order to increase the activity of the wild type Geobacillus thermocatenulatus lipase 2 (BTL2) we rationally designed, based on its tridimensional structure, a mutant (ccBTL2) capable of forming a disulfide bond to lock the open state. ccBTL2 was generated replacing A191 and F206 to cysteine residues while both wild type C64 and C295 were mutated to serine. A covalently immobilized ccBTL2 showed a 3.5-fold increment in esterase activity with 0.1% Triton X-100 (2336 IU mg-1) and up to 6.0-fold higher with 0.01% CTAB (778 IU mg-1), both in the presence of oxidizing sulfhydryl agents, when compared to BTL2. The remarkable and industrially desired features of BTL2 such as optimal alkaliphilic pH and high thermal stability were not affected. The designed disulfide bond also conferred reversibility to the enhancement, as the increment on activity observed for ccBTL2 was controlled by redox pretreatments. MD simulations suggested that the most stable conformation for ccBTL2 (with the disulfide bond formed) was, as we predicted, similar to the open and active conformation of this lipase.

How allosteric control of Staphylococcus aureus penicillin binding protein 2a enables methicillin resistance and physiological function.

The expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical resistance to the ß-lactam antibiotics by methicillin-resistant Staphylococcus aureus (MRSA). The high-molecular mass penicillin binding proteins of bacteria catalyze in separate domains the transglycosylase and transpeptidase activities required for the biosynthesis of the peptidoglycan polymer that comprises the bacterial cell wall. In bacteria susceptible to ß-lactam antibiotics, the transpeptidase activity of their penicillin binding proteins (PBPs) is lost as a result of irreversible acylation of an active site serine by the ß-lactam antibiotics. In contrast, the PBP2a of MRSA is resistant to ß-lactam acylation and successfully catalyzes the DD-transpeptidation reaction necessary to complete the cell wall. The inability to contain MRSA infection with ß-lactam antibiotics is a continuing public health concern. We report herein the identification of an allosteric binding domain--a remarkable 60 Å distant from the DD-transpeptidase active site--discovered by crystallographic analysis of a soluble construct of PBP2a. When this allosteric site is occupied, a multiresidue conformational change culminates in the opening of the active site to permit substrate entry. This same crystallographic analysis also reveals the identity of three allosteric ligands: muramic acid (a saccharide component of the peptidoglycan), the cell wall peptidoglycan, and ceftaroline, a recently approved anti-MRSA ß-lactam antibiotic. The ability of an anti-MRSA ß-lactam antibiotic to stimulate allosteric opening of the active site, thus predisposing PBP2a to inactivation by a second ß-lactam molecule, opens an unprecedented realm for ß-lactam antibiotic structure-based design.

Structural basis for the broad specificity of a new family of amino-acid racemases.

Broad-spectrum amino-acid racemases (Bsrs) enable bacteria to generate noncanonical D-amino acids, the roles of which in microbial physiology, including the modulation of cell-wall structure and the dissolution of biofilms, are just beginning to be appreciated. Here, extensive crystallographic, mutational, biochemical and bioinformatic studies were used to define the molecular features of the racemase BsrV that enable this enzyme to accommodate more diverse substrates than the related PLP-dependent alanine racemases. Conserved residues were identified that distinguish BsrV and a newly defined family of broad-spectrum racemases from alanine racemases, and these residues were found to be key mediators of the multispecificity of BrsV. Finally, the structural analysis of an additional Bsr that was identified in the bioinformatic analysis confirmed that the distinguishing features of BrsV are conserved among Bsr family members.

Disruption of allosteric response as an unprecedented mechanism of resistance to antibiotics.

Ceftaroline, a recently approved ß-lactam antibiotic for treatment of infections by methicillin-resistant Staphylococcus aureus (MRSA), is able to inhibit penicillin-binding protein 2a (PBP2a) by triggering an allosteric conformational change that leads to the opening of the active site. The opened active site is now vulnerable to inhibition by a second molecule of ceftaroline, an event that impairs cell-wall biosynthesis and leads to bacterial death. The triggering of the allosteric effect takes place by binding of the first antibiotic molecule 60 Å away from the active site of PBP2a within the core of the allosteric site. We document, by kinetic studies and by determination of three X-ray structures of the mutant variants of PBP2a that result in resistance to ceftaroline, that the effect of these clinical mutants is the disruption of the allosteric trigger in this important protein in MRSA. This is an unprecedented mechanism for antibiotic resistance.

Structural characterization of PaaX, the main repressor of the phenylacetate degradation pathway in Escherichia coli W: A novel fold of transcription regulator proteins.

PaaX is a transcriptional repressor of the phenylacetic acid (PAA) catabolic pathway, a central route for bacterial aerobic degradation of aromatic compounds. Induction of the route is achieved through the release of PaaX from its promoter sequences by the first compound of the pathway, phenylacetyl-coenzyme A (PA-CoA). We report the crystal structure of PaaX from Escherichia coli W. PaaX displays a novel type of fold for transcription regulators, showing a dimeric conformation where the monomers present a three-domain structure: an N-terminal winged helix-turn-helix domain, a dimerization domain similar to the Cas2 protein and a C-terminal domain without structural homologs. The domains are separated by a crevice amenable to harbour a PA-CoA molecule. The biophysical characterization of the protein in solution confirmed several hints predicted from the structure, i.e. its dimeric conformation, a modest importance of cysteines and a high dependence of solubility and thermostability on ionic strength. At a moderately acidic pH, the protein formed a stable folding intermediate with remaining α-helical structure, a disrupted tertiary structure and exposed hydrophobic patches. Our results provide valuable information to understand the stability and mechanism of PaaX and pave the way for further analysis of other regulators with similar structural configurations.

Cell-wall remodeling by the zinc-protease AmpDh3 from Pseudomonas aeruginosa.

Bacterial cell wall is a polymer of considerable complexity that is in constant equilibrium between synthesis and recycling. AmpDh3 is a periplasmic zinc protease of Pseudomonas aeruginosa , which is intimately involved in cell-wall remodeling. We document the hydrolytic reactions that this enzyme performs on the cell wall. The process removes the peptide stems from the peptidoglycan, the major constituent of the cell wall. We document that the majority of the reactions of this enzyme takes place on the polymeric insoluble portion of the cell wall, as opposed to the fraction that is released from it. We show that AmpDh3 is tetrameric both in crystals and in solution. Based on the X-ray structures of the enzyme in complex with two synthetic cell-wall-based ligands, we present for the first time a model for a multivalent anchoring of AmpDh3 onto the cell wall, which lends itself to its processive remodeling.

Reaction products and the X-ray structure of AmpDh2, a virulence determinant of Pseudomonas aeruginosa.

The zinc protease AmpDh2 is a virulence determinant of Pseudomonas aeruginosa, a problematic human pathogen. The mechanism of how the protease manifests virulence is not known, but it is known that it turns over the bacterial cell wall. The reaction of AmpDh2 with the cell wall was investigated, and nine distinct turnover products were characterized by LC/MS/MS. The enzyme turns over both the cross-linked and noncross-linked cell wall. Three high-resolution X-ray structures, the apo enzyme and two complexes with turnover products, were solved. The X-ray structures show how the dimeric protein interacts with the inner leaflet of the bacterial outer membrane and that the two monomers provide a more expansive surface for recognition of the cell wall. This binding surface can accommodate the 3D solution structure of the cross-linked cell wall.

Crystal structures of bacterial peptidoglycan amidase AmpD and an unprecedented activation mechanism.

AmpD is a cytoplasmic peptidoglycan (PG) amidase involved in bacterial cell-wall recycling and in induction of ß-lactamase, a key enzyme of ß-lactam antibiotic resistance. AmpD belongs to the amidase_2 family that includes zinc-dependent amidases and the peptidoglycan-recognition proteins (PGRPs), highly conserved pattern-recognition molecules of the immune system. Crystal structures of Citrobacter freundii AmpD were solved in this study for the apoenzyme, for the holoenzyme at two different pH values, and for the complex with the reaction products, providing insights into the PG recognition and the catalytic process. These structures are significantly different compared with the previously reported NMR structure for the same protein. The NMR structure does not possess an accessible active site and shows the protein in what is proposed herein as an inactive "closed" conformation. The transition of the protein from this inactive conformation to the active "open" conformation, as seen in the x-ray structures, was studied by targeted molecular dynamics simulations, which revealed large conformational rearrangements (as much as 17 Å) in four specific regions representing one-third of the entire protein. It is proposed that the large conformational change that would take the inactive NMR structure to the active x-ray structure represents an unprecedented mechanism for activation of AmpD. Analysis is presented to argue that this activation mechanism might be representative of a regulatory process for other intracellular members of the bacterial amidase_2 family of enzymes.

Biosensor for branched-chain amino acid metabolism in yeast and applications in isobutanol and isopentanol production.

Branched-chain amino acid (BCAA) metabolism fulfills numerous physiological roles and can be harnessed to produce valuable chemicals. However, the lack of eukaryotic biosensors specific for BCAA-derived products has limited the ability to develop high-throughput screens for strain engineering and metabolic studies. Here, we harness the transcriptional regulator Leu3p from Saccharomyces cerevisiae to develop a genetically encoded biosensor for BCAA metabolism. In one configuration, we use the biosensor to monitor yeast production of isobutanol, an alcohol derived from valine degradation. Small modifications allow us to redeploy Leu3p in another biosensor configuration that monitors production of the leucine-derived alcohol, isopentanol. These biosensor configurations are effective at isolating high-producing strains and identifying enzymes with enhanced activity from screens for branched-chain higher alcohol (BCHA) biosynthesis in mitochondria as well as cytosol. Furthermore, this biosensor has the potential to assist in metabolic studies involving BCAA pathways, and offers a blueprint to develop biosensors for other products derived from BCAA metabolism.

High-resolution crystal structure of MltE, an outer membrane-anchored endolytic peptidoglycan lytic transglycosylase from Escherichia coli.

The crystal structure of the first endolytic peptidoglycan lytic transglycosylase MltE from Escherichia coli is reported here. The degradative activity of this enzyme initiates the process of cell wall recycling, which is an integral event in the existence of bacteria. The structure sheds light on how MltE recognizes its substrate, the cell wall peptidoglycan. It also explains the ability of this endolytic enzyme to cleave in the middle of the peptidoglycan chains. Furthermore, the structure reveals how the enzyme is sequestered on the inner leaflet of the outer membrane.

Crystallization and preliminary X-ray diffraction studies of the transcriptional repressor PaaX, the main regulator of the phenylacetic acid degradation pathway in Escherichia coli W.

PaaX is the main regulator of the phenylacetic acid aerobic degradation pathway in bacteria and acts as a transcriptional repressor in the absence of its inducer phenylacetyl-coenzyme A. The natural presence and the recent accumulation of a variety of highly toxic aromatic compounds owing to human pollution has created considerable interest in the study of degradation pathways in bacteria, the most important microorganisms capable of recycling these compounds, in order to design and apply novel bioremediation strategies. PaaX from Escherichia coli W was cloned, overexpressed, purified and crystallized using the sitting-drop vapour-diffusion method at 291 K. Crystals grew from a mixture of 0.9 M Li(2)SO(4) and 0.5 M sodium citrate pH 5.8. These crystals, which belonged to the monoclinic space group C2 with unit-cell parameters a = 167.88, b = 106.23, c = 85.87 Å, ß = 108.33°, allowed the collection of an X-ray data set to 2.3 Å resolution.

The elusive relationship between structure and colour emission in beetle luciferases.

In beetles, luciferase enzymes catalyse the conversion of chemical energy into light through bioluminescence. The principles of this process have become a fundamental biotechnological tool that revolutionized biological research. Different beetle species can emit different colours of light, despite using the same substrate and highly homologous luciferases. The chemical reasons for these different colours are hotly debated yet remain unresolved. This Review summarizes the structural, biochemical and spectrochemical data on beetle bioluminescence reported over the past three decades. We identify the factors that govern what colour is emitted by wild-type and mutant luciferases. This topic is controversial, but, in general, we note that green emission requires cationic residues in a specific position near the benzothiazole fragment of the emitting molecule, oxyluciferin. The commonly emitted green-yellow light can be readily changed to red by introducing a variety of individual and multiple mutations. However, complete switching of the emitted light from red to green has not been accomplished and the synergistic effects of combined mutations remain unexplored. The minor colour shifts produced by most known mutations could be important in establishing a 'mutational catalogue' to fine-tune emission of beetle luciferases, thereby expanding the scope of their applications.

Lights up on organelles: Optogenetic tools to control subcellular structure and organization.

Since the neurobiological inception of optogenetics, light-controlled molecular perturbations have been applied in many scientific disciplines to both manipulate and observe cellular function. Proteins exhibiting light-sensitive conformational changes provide researchers with avenues for spatiotemporal control over the cellular environment and serve as valuable alternatives to chemically inducible systems. Optogenetic approaches have been developed to target proteins to specific subcellular compartments, allowing for the manipulation of nuclear translocation and plasma membrane morphology. Additionally, these tools have been harnessed for molecular interrogation of organelle function, location, and dynamics. Optogenetic approaches offer novel ways to answer fundamental biological questions and to improve the efficiency of bioengineered cell factories by controlling the assembly of synthetic organelles. This review first provides a summary of available optogenetic systems with an emphasis on their organelle-specific utility. It then explores the strategies employed for organelle targeting and concludes by discussing our perspective on the future of optogenetics to control subcellular structure and organization. This article is categorized under: Metabolic Diseases > Molecular and Cellular Physiology.

Binding of non-canonical peptidoglycan controls Vibrio cholerae broad spectrum racemase activity.

Broad-spectrum amino acid racemases (Bsrs) enable bacteria to generate non-canonical D-amino acids (NCDAAs), whose roles and impact on microbial physiology, including modulation of cell wall structure and dissolution of biofilms, are just beginning to be appreciated. Here we used a diverse array of structural, biochemical and molecular simulation studies to define and characterize how BsrV is post-translationally regulated. We discovered that contrary to Vibrio cholerae alanine racemase AlrV highly compacted active site, BsrV's is broader and can be occupied by cell wall stem peptides. We found that peptidoglycan peptides modified with NCDAAs are better stabilized by BsrV's catalytic cavity and show better inhibitory capacity than canonical muropeptides. Notably, BsrV binding and inhibition can be recapitulated by undigested peptidoglycan sacculi as it exists in the cell. Docking simulations of BsrV binding the peptidoglycan polymer generate a model where the peptide stems are perfectly accommodated and stabilized within each of the dimers active sites. Taking these biochemical and structural data together, we propose that inhibition of BsrV by peptidoglycan peptides underlies a negative regulatory mechanism to avoid excessive NCDAA production. Our results collectively open the door to use "à la carte" synthetic peptides as a tool to modulate DAAs production of Bsr enzymes.

Optogenetics and biosensors set the stage for metabolic cybergenetics.

Cybergenetic systems use computer interfaces to enable feed-back controls over biological processes in real time. The complex and dynamic nature of cellular metabolism makes cybergenetics attractive for controlling engineered metabolic pathways in microbial fermentations. Cybergenetics would not only create new avenues of research into cellular metabolism, it would also enable unprecedented strategies for pathway optimization and bioreactor operation and automation. Implementation of metabolic cybergenetics, however, will require new capabilities from actuators, biosensors, and control algorithms. The recent application of optogenetics in metabolic engineering, the expanding role of genetically encoded biosensors in strain development, and continued progress in control algorithms for biological processes suggest that this technology will become available in the not so distant future.

Development of light-responsive protein binding in the monobody non-immunoglobulin scaffold.

Monobodies are synthetic non-immunoglobulin customizable protein binders invaluable to basic and applied research, and of considerable potential as future therapeutics and diagnostic tools. The ability to reversibly control their binding activity to their targets on demand would significantly expand their applications in biotechnology, medicine, and research. Here we present, as proof-of-principle, the development of a light-controlled monobody (OptoMB) that works in vitro and in cells and whose affinity for its SH2-domain target exhibits a 330-fold shift in binding affinity upon illumination. We demonstrate that our αSH2-OptoMB can be used to purify SH2-tagged proteins directly from crude E. coli extract, achieving 99.8% purity and over 40% yield in a single purification step. By virtue of their ability to be designed to bind any protein of interest, OptoMBs have the potential to find new powerful applications as light-switchable binders of untagged proteins with the temporal and spatial precision afforded by light.

Optogenetic control of protein binding using light-switchable nanobodies.

A growing number of optogenetic tools have been developed to reversibly control binding between two engineered protein domains. In contrast, relatively few tools confer light-switchable binding to a generic target protein of interest. Such a capability would offer substantial advantages, enabling photoswitchable binding to endogenous target proteins in cells or light-based protein purification in vitro. Here, we report the development of opto-nanobodies (OptoNBs), a versatile class of chimeric photoswitchable proteins whose binding to proteins of interest can be enhanced or inhibited upon blue light illumination. We find that OptoNBs are suitable for a range of applications including reversibly binding to endogenous intracellular targets, modulating signaling pathway activity, and controlling binding to purified protein targets in vitro. This work represents a step towards programmable photoswitchable regulation of a wide variety of target proteins.