Studies on selected enzymes of the alpha-aminoadipate pathway for lysine biosynthesis in Saccharomyces cerevisiae.

Homocitrate synthase (HCS) catalyzes one of the regulated steps of the alpha-aminoadipate pathway for Lys biosynthesis in fungi. His-tagged HCS from Saccharomyces cerevisiae was purified to about 98% using a Ni-NTA resin and stabilized. The enzyme, in the absence of reactants, self associates, as suggested by size exclusion chromatography. Fluorescence and circular dichroic spectra suggested a partially exposed Trp residue and a mixed (alpha/beta) secondary structure for the enzyme. Fluorescence quenching studies with KI, CsCl, and acrylamide, suggest that the microenvironment around the single Trp residue of the enzyme has some positive charge.The kinetic mechanism of regulation of HCS from S. cerevisiae by Na+ and the feedback inhibitor Lys was studied by measuring the initial rate in the absence and presence of the effectors. Data suggest that Na+ is an activator at low concentrations and an inhibitor at high concentrations, and these effects occur as a result of the monovalent ion binding to two different sites in the free enzyme. Inhibition and activation by Na+ can occur simultaneously. The inhibition by Na+ is eliminated at high concentrations of AcCoA, the second substrate bound, but the activation remains. Fluorescence binding studies indicate Lys binds with high affinity to its binding site as an inhibitor. The inhibition by Lys is competitive and linear vs. alpha-Kg. The effects of Na+ and Lys are independent of one another. A model is developed for regulation of HCS that takes into account all of the effects discussed above. The rate equations that predict the regulatory kinetic behavior of HCS are also derived, and simulation of the predicted behavior is carried out over a range of values for the kinetic parameters. (Abstract shortened by UMI.)Kinetic data have been collected suggesting a preferred sequential ordered kinetic mechanism for the His-tagged HCS from S. cerevisiae with alpha-Kg binding before AcCoA and CoA released before Hc. In agreement with the ordered kinetic mechanism desulfo-CoA is uncompetitive and citrate is competitive vs. alpha-Kg. Varying AcCoA, citrate is a noncompetitive inhibitor as predicted, but CoA is noncompetitive vs. AcCoA suggesting binding of CoA to E:Hc and E:alpha-Kg. The product CoA behaves identical to desulfo-CoA, suggesting an E:alpha-Kg:CoA dead-end complex. Data further suggest an irreversible reaction overall. Fluorescence titration data show finite binding of CoA and AcCoA to free enzyme, suggesting the mechanism may be random with a high degree of synergism of binding between the reactants

Crystallographic and Spectroscopic Snapshots Reveal a Dehydrogenase in Action

Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for ‘disarming’ the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacyl intermediate and an NADþ-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp3-to-sp2 transition during enzyme-mediated oxidation

The Structure Of The Giant Haemoglobin From Glossoscolex Paulistus.

The sequences of all seven polypeptide chains from the giant haemoglobin of the free-living earthworm Glossoscolex paulistus (HbGp) are reported together with the three-dimensional structure of the 3.6 MDa complex which they form. The refinement of the full particle, which has been solved at 3.2 Å resolution, the highest resolution reported to date for a hexagonal bilayer haemoglobin composed of 12 protomers, is reported. This has allowed a more detailed description of the contacts between subunits which are essential for particle stability. Interpretation of features in the electron-density maps suggests the presence of metal-binding sites (probably Zn(2+) and Ca(2+)) and glycosylation sites, some of which have not been reported previously. The former appear to be important for the integrity of the particle. The crystal structure of the isolated d chain (d-HbGp) at 2.1 Å resolution shows different interchain contacts between d monomers compared with those observed in the full particle. Instead of forming trimers, as seen in the complex, the isolated d chains associate to form dimers across a crystallographic twofold axis. These observations eliminate the possibility that trimers form spontaneously in solution as intermediates during the formation of the dodecameric globin cap and contribute to understanding of the possible ways in which the particle self-assembles.711257-127

The temperature-dependent conformational ensemble of SARS-CoV-2 main protease (Mpro)

The COVID-19 pandemic, instigated by the SARS-CoV-2 coronavirus, continues to plague the globe. The SARS-CoV-2 main protease, or Mpro, is a promising target for development of novel antiviral therapeutics. Previous X-ray crystal structures of Mpro were obtained at cryogenic temperature or room temperature only. Here we report a series of high-resolution crystal structures of unliganded Mpro across multiple temperatures from cryogenic to physiological, and another at high humidity. We interrogate these datasets with parsimonious multiconformer models, multi-copy ensemble models, and isomorphous difference density maps. Our analysis reveals a temperature-dependent conformational landscape for Mpro, including mobile solvent interleaved between the catalytic dyad, mercurial conformational heterogeneity in a key substrate-binding loop, and a far-reaching intramolecular network bridging the active site and dimer interface. Our results may inspire new strategies for antiviral drug development to counter-punch COVID-19 and combat future coronavirus pandemics

Acoustic Injectors For Drop-On-Demand Serial Femtosecond Crystallography

X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples

AlphaFold Protein Structure Database for Sequence-Independent Molecular Replacement

Crystallographic phasing recovers the phase information that is lost during a diffraction experiment. Molecular replacement is a commonly used phasing method for crystal structures in the protein data bank. In one form it uses a protein sequence to search a structure database to find suitable templates for phasing. However, sequence information is not always available, such as when proteins are crystallized with unknown binding partner proteins or when the crystal is of a contaminant. The recent development of AlphaFold published the predicted protein structures for every protein from twenty distinct species. In this work, we tested whether AlphaFold-predicted E. coli protein structures were accurate enough to enable sequence-independent phasing of diffraction data from two crystallization contaminants of unknown sequence. Using each of more than 4000 predicted structures as a search model, robust molecular replacement solutions were obtained, which allowed the identification and structure determination of YncE and YadF. Our results demonstrate the general utility of the AlphaFold-predicted structure database with respect to sequence-independent crystallographic phasing

Hepatitis C virus NS3/4A inhibitors and other drug-like compounds as covalent binders of SARS-CoV-2 main protease.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), threatens global public health. The world needs rapid development of new antivirals and vaccines to control the current pandemic and to control the spread of the variants. Among the proteins synthesized by the SARS-CoV-2 genome, main protease (Mpro also known as 3CLpro) is a primary drug target, due to its essential role in maturation of the viral polyproteins. In this study, we provide crystallographic evidence, along with some binding assay data, that three clinically approved anti hepatitis C virus drugs and two other drug-like compounds covalently bind to the Mpro Cys145 catalytic residue in the active site. Also, molecular docking studies can provide additional insight for the design of new antiviral inhibitors for SARS-CoV-2 using these drugs as lead compounds. One might consider derivatives of these lead compounds with higher affinity to the Mpro as potential COVID-19 therapeutics for further testing and possibly clinical trials