In nature as in biotechnology, light-oxygen-voltage photoreceptors perceive blue light to elicit spatiotemporally defined cellular responses. Photon absorption drives thioadduct formation between a conserved cysteine and the flavin chromophore. An equally conserved, proximal glutamine processes the resultant flavin protonation into downstream hydrogen-bond rearrangements. Here, we report that this glutamine, long deemed essential, is generally dispensable. In its absence, several light-oxygen-voltage receptors invariably retained productive, if often attenuated, signaling responses. Structures of a light-oxygen-voltage paradigm at around 1 Å resolution revealed highly similar light-induced conformational changes, irrespective of whether the glutamine is present. Naturally occurring, glutamine-deficient light-oxygen-voltage receptors likely serve as bona fide photoreceptors, as we showcase for a diguanylate cyclase. We propose that without the glutamine, water molecules transiently approach the chromophore and thus propagate flavin protonation downstream. Signaling without glutamine appears intrinsic to light-oxygen-voltage receptors, which pertains to biotechnological applications and suggests evolutionary descendance from redox-active flavoproteins.

The enzyme glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first step of the oxidative branch of the pentose phosphate pathway, which provides cells with NADPH, an essential cofactor for many biosynthetic pathways and antioxidizing enzymes. In Trypanosoma cruzi, the G6PDH has being pursued as a relevant target for the development of new drugs against Chagas disease. At present, the best characterized inhibitors of T. cruzi G6PDH are steroidal halogenated compounds derivatives from the mammalian hormone precursor dehydroepiandrosterone, which indeed are also good inhibitors of the human homologue enzyme. The lack of target selectivity might result in hemolytic side effects due to partial inhibition of human G6PDH in red blood cells. Moreover, the treatment of Chagas patients with steroidal drugs might also cause undesired androgenic side effects. Aiming to identify of new chemical classes of T. cruzi G6PDH inhibitors, we performed a target-based high-throughput screen campaign against a commercial library of diverse compounds. Novel TcG6PDH inhibitors were identified among thienopyrimidine and quinazolinone derivatives. Preliminary structure activity relationships for the identified hits are presented, including structural features that contribute for selectivity toward the parasite enzyme. Our results indicate that quinazolinones are promising hits that should be considered for further optimization.

Trypanosoma cruzi is the causative agent of Chagas disease. The lack of an efficient and safe treatment supports the research into novel metabolic targets, with the malic enzyme (ME) representing one such potential candidate. T. cruzi expresses a cytosolic (TcMEc) and a mitochondrial (TcMEm) ME isoform, with these activities functioning to generate NADPH, a key source of reducing equivalents that drives a range of anabolic and protective processes. To identify specific inhibitors that target TcMEs, two independent high-throughput screening strategies using a diversity library containing 30,000 compounds were employed. IC values of 262 molecules were determined for both TcMEs, as well as for three human ME isoforms, with the inhibitors clustered into six groups according to their chemical similarity. The most potent hits belonged to a sulfonamide group that specifically target TcMEc. Moreover, several selected inhibitors of both TcMEs showed a trypanocidal effect against the replicative forms of T. cruzi. The chemical diversity observed among those compounds that inhibit TcMEs activity emphasizes the druggability of these enzymes, with a sulfonamide-based subset of compounds readily able to block TcMEc function at a low nanomolar range.

Ubiquitin-conjugating enzymes (E2) enable protein ubiquitination by conjugating ubiquitin to their catalytic cysteine for subsequent transfer to a target lysine sidechain. Deprotonation of the incoming lysine enables its nucleophilicity, but determinants of lysine activation remain poorly understood. We report a novel pathogenic mutation in the E2 UBE2A, identified in two brothers presenting mild intellectual disability. The pathogenic Q93E mutation yields UBE2A with impaired aminolysis activity but no loss of ability to be conjugated with ubiquitin. Importantly, the low intrinsic reactivity of Q93E-UBE2A is not overcome by a cognate ubiquitin E3 ligase, Rad18, with the UBE2A target, PCNA. However, Q93E-UBE2A is reactive at high pH or with a low pKa amine as the nucleophile, providing the first evidence of reversion of a defective UBE2A mutation. We propose that Q93E substitution perturbs the UBE2A catalytic microenvironment essential for lysine deprotonation during ubiquitin transfer, generating an enzyme that is disabled but not dead.

In optogenetics, as in nature, sensory photoreceptors serve to control cellular processes by light. Bacteriophytochrome (BphP) photoreceptors sense red and farred light via a biliverdin chromophore and, in response, cycle between the spectroscopically, structurally, and functionally distinct Pr and Pfr states. BphPs commonly belong to two-component systems that control the phosphorylation of cognate response regulators and downstream gene expression through histidine kinase modules. We recently demonstrated that the paradigm BphP from Deinococcus radiodurans exclusively acts as a phosphatase but that its photosensory module can control the histidine kinase activity of homologous receptors. Here, we apply this insight to reprogram two widely used setups for bacterial gene expression from blue-light to red-light control. The resultant pREDusk and pREDawn systems allow gene expression to be regulated down and up, respectively, uniformly under red light by 100-fold or more. Both setups are realized as portable, single plasmids that encode all necessary components including the biliverdin-producing machinery. The triggering by red light affords high spatial resolution down to the single-cell level. As pREDusk and pREDawn respond sensitively to red light, they support multiplexing with optogenetic systems sensitive to other light colors. Owing to the superior tissue penetration of red light, the pREDawn system can be triggered at therapeutically safe light intensities through material layers, replicating the optical properties of the skin and skull. Given these advantages, pREDusk and pREDawn enable red-light-regulated expression for diverse use cases in bacteria.

Sensory photoreceptors mediate numerous light-dependent adaptations across organisms. In optogenetics, photoreceptors achieve the reversible, non-invasive, and spatiotemporally precise control by light of gene expression and other cellular processes. The light-oxygen-voltage receptor PAL binds to small RNA aptamers with sequence specificity upon blue-light illumination. By embedding the responsive aptamer in the ribosome-binding sequence of genes of interest, their expression can be downregulated by light. We developed the pCrepusculo and pAurora optogenetic systems that are based on PAL and allow to down-and upregulate, respectively, bacterial gene expression using blue light. Both systems are realized as compact, single plasmids that exhibit stringent blue-light responses with low basal activity and up to several 10-fold dynamic range. As PAL exerts light-dependent control at the RNA level, it can be combined with other optogenetic circuits that control transcription initiation. By integrating regulatory mechanisms operating at the DNA and mRNA levels, optogenetic circuits with emergent properties can thus be devised. As a case in point, the pEnumbra setup permits to upregulate gene expression under moderate blue light whereas strong blue light shuts off expression again. Beyond providing novel signal-responsive expression systems for diverse applications in biotechnology and synthetic biology, our work also illustrates how the light-dependent PAL-aptamer interaction can be harnessed for the control and interrogation of RNA-based processes.

Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the oxidation of glucose-6-phoshate to 6-phospho-gluconolactone with the concomitant reduction of NADP to NADPH. In solution, the recombinant human G6PDH is known to be active as dimers and tetramers. To distinguish between the kinetic properties of dimers and tetramers of the G6PDH is not trivial. Steady-state kinetic experiments are often performed at low enzyme concentrations, which favor the dimeric state. The present work describes two novel human G6PDH mutants, one that creates four disulfide bonds among apposing dimers, resulting in a 'cross-linked' tetramer, and another that prevents the dimer to dimer association. The functional and structural characterizations of such mutants indicate the tetramer as the most active form of human G6PDH.

To shed more light on the molecular requirements for recognition of thyroid response elements (TREs) by thyroid receptors (TRs), we compared the specific aspects of DNA TRE recognition by different TR constructs. Using fluorescence anisotropy, we performed a detailed and hierarchical study of TR-TRE binding. This was done by comparing the binding affinities of three different TR constructs for four different TRE DNA elements, including palindromic sequences and direct repeats (F2, PAL, DR-1, and DR-4) as well as their interactions with nonspecific DNA sequences. The effect of MgCl(2) on suppressing of nonselective DNA binding to TR was also investigated. Furthermore, we determined the dissociation constants of the hTRbeta DBD (DNA binding domain) and hTRbeta DBD-LBD (DNA binding and ligand binding domains) for specific TREs. We found that a minimum DNA recognition peptide derived from DBD (H1TR) is sufficient for recognition and interaction with TREs, whereas scrambled DNA sequences were unrecognized. Additionally, we determined that the TR DBD binds to F2, PAL, and DR-4 with high affinity and similar K(d) values. The TR DBD-LBD recognizes all the tested TREs but binds preferentially to F2, with even higher affinity. Finally, our results demonstrate the important role played by LBDs in modulating TR-DNA binding.