Crystallization of 3-hexulose-6-phosphate synthase

The crystal structures can reveal detailed information about the overall structure, active site structure, and functional mechanism of enzymes. This study focused on the crystallization of 3-hexulose-6-phosphate synthase from Methylomonas aminofaciens 77a, to produce higher resolution crystals for precise structural characterization. 3-Hexulose-6-phosphate synthase is from Methylomonas aminofaciens 77a (EC 4.1.2.43). It belongs to the orotidine 5'-monophosphate decarboxylase superfamily, and acts as a key enzyme for a ribulose-monophosphate cycle of formaldehyde fixation and detoxification. 3-Hexulose-6-phosphate synthase catalyzes the aldol condensation of formaldehyde with D-ribulose-5-phosphate. For the maximum activity, 3-hexulose-6-phosphate synthase requires Mg2+ or Mn2+ as ligands. MaHPS crystallized at the concentration of 7 mg/mL and conditions consisting of 0.2 M MgCl2, 18% PEG 3350 at pH = 7.0.

Iron-Imprinted Single-Atomic Site Catalyst-Based Nanoprobe for Detection of Hydrogen Peroxide in Living Cells

Fe-based single-atomic site catalysts (SASCs), with the natural metalloproteases-like active site structure, have attracted widespread attention in biocatalysis and biosensing. Precisely, controlling the isolated single-atom Fe-N-C active site structure is crucial to improve the SASCs’ performance. In this work, we use a facile ion-imprinting method (IIM) to synthesize isolated Fe-N-C single-atomic site catalysts (IIM-Fe-SASC). With this method, the ion-imprinting process can precisely control ion at the atomic level and form numerous well-defined single-atomic Fe-N-C sites. The IIM-Fe-SASC shows better peroxidase-like activities than that of non-imprinted references. Due to its excellent properties, IIM-Fe-SASC is an ideal nanoprobe used in the colorimetric biosensing of hydrogen peroxide (H2O2). Using IIM-Fe-SASC as the nanoprobe, in situ detection of H2O2 generated from MDA-MB-231 cells has been successfully demonstrated with satisfactory sensitivity and specificity. This work opens a novel and easy route in designing advanced SASC and provides a sensitive tool for intracellular H2O2 detection.

Highly selective and robust single-atom catalyst Ru1/NC for reductive amination of aldehydes/ketones

Single-atom catalysts (SACs) have emerged as a frontier in heterogeneous catalysis due to the well-defined active site structure and the maximized metal atom utilization. Nevertheless, the robustness of SACs remains a critical concern for practical applications. Herein, we report a highly active, selective and robust Ru SAC which was synthesized by pyrolysis of ruthenium acetylacetonate and N/C precursors at 900 °C in N2 followed by treatment at 800 °C in NH3. The resultant Ru1-N3 structure exhibits moderate capability for hydrogen activation even in excess NH3, which enables the effective modulation between transimination and hydrogenation activity in the reductive amination of aldehydes/ketones towards primary amines. As a consequence, it shows superior amine productivity, unrivalled resistance against CO and sulfur, and unexpectedly high stability under harsh hydrotreating conditions compared to most SACs and nanocatalysts. This SAC strategy will open an avenue towards the rational design of highly selective and robust catalysts for other demanding transformations.

Neutron crystallography of copper amine oxidase reveals keto/enolate interconversion of the quinone cofactor and unusual proton sharing

Recent advances in neutron crystallographic studies have provided structural bases for quantum behaviors of protons observed in enzymatic reactions. Thus, we resolved the neutron crystal structure of a bacterial copper (Cu) amine oxidase (CAO), which contains a prosthetic Cu ion and a protein-derived redox cofactor, topa quinone (TPQ). We solved hitherto unknown structures of the active site, including a keto/enolate equilibrium of the cofactor with a nonplanar quinone ring, unusual proton sharing between the cofactor and the catalytic base, and metal-induced deprotonation of a histidine residue that coordinates to the Cu. Our findings show a refined active-site structure that gives detailed information on the protonation state of dissociable groups, such as the quinone cofactor, which are critical for catalytic reactions.

Copper-only superoxide dismutase enzymes and iron starvation stress in Candida fungal pathogens

Copper (Cu)-only superoxide dismutases (SOD) represent a newly characterized class of extracellular SODs important for virulence of several fungal pathogens. Previous studies of the Cu-only enzyme SOD5 from the opportunistic fungal pathogen Candida albicans have revealed that the active-site structure and Cu binding of SOD5 strongly deviate from those of Cu/Zn-SODs in its animal hosts, making Cu-only SODs a possible target for future antifungal drug design. C. albicans also expresses a Cu-only SOD4 that is highly similar in sequence to SOD5, but is poorly characterized. Here, we compared the biochemical, biophysical, and cell biological properties of C. albicans SOD4 and SOD5. Analyzing the recombinant proteins, we found that, similar to SOD5, Cu-only SOD4 can react with superoxide at rates approaching diffusion limits. Both SODs were monomeric and they exhibited similar binding affinities for their Cu cofactor. In C. albicans cultures, SOD4 and SOD5 were predominantly cell wall proteins. Despite these similarities, the SOD4 and SOD5 genes strongly differed in transcriptional regulation. SOD5 was predominantly induced during hyphal morphogenesis, together with a fungal burst in reactive oxygen species. Conversely, SOD4 expression was specifically up-regulated by iron (Fe) starvation and controlled by the Fe-responsive transcription factor SEF1. Interestingly, Candida tropicalis and the emerging fungal pathogen Candida auris contain a single SOD5-like SOD rather than a pair, and in both fungi, this SOD was induced by Fe starvation. This unexpected link between Fe homeostasis and extracellular Cu-SODs may help many fungi adapt to Fe-limited conditions of their hosts.