A Host-Guest Approach to Engineering Oxidase-Mimetic Assembly with Substrate Selectivity and Dynamic Catalysis.

The creation of synthetic materials that emulate the complexity of natural systems, such as enzymes, remains a challenge in biomimicry. Here, we present a simple yet effective strategy to introduce substrate selectivity and dynamic responsiveness into an enzyme-mimetic supramolecular material. We achieved this by anchoring γ-cyclodextrin to a fluorene-modified Lys/Cu2+ assembly, which mimics copper-dependent oxidase. The binding affinity among the components was examined using 1H NMR, isothermal titration calorimetry (ITC), and theoretical simulation. The γ-cyclodextrin acts as a host, forming a complex with the fluorenyl moiety and aromatic substrates of specific sizes. This ensures the proximity of the substrate reactive groups to the copper center, leading to size-selective enhancement of aromatic substrate oxidation, particularly favoring biphenyl substrates. Notably, α- and ß-cyclodextrins do not exhibit this effect, and the native oxidase lacks this selectivity. Additionally, the binding affinity of the aromatic substrate to the catalyst can be dynamically tuned by adding α-cyclodextrin or by irradiating with different wavelengths in the presence of competitive azo-guests, resulting in switched oxidative activities. This approach offers a new avenue for designing biomimetic materials with tailorable active site structures and catalytic properties.

Functional Characterization of the Lysosomal Peptide/Histidine Transporter PHT1 (SLC15A4) by Solid Supported Membrane Electrophysiology (SSME).

The peptide/histidine transporter PHT1 (SLC15A4) is expressed in the lysosomal membranes of immune cells where it plays an important role in metabolic and inflammatory signaling. PHT1 is an H+-coupled/histidine symporter that can transport a wide range of oligopeptides, including a variety of bacterial-derived peptides. Moreover, it enables the scaffolding of various metabolic signaling molecules and interacts with key regulatory elements of the immune response. Not surprisingly, PHT1 has been implicated in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). Unfortunately, the pharmacological development of PHT1 modulators has been hampered by the lack of suitable transport assays. To address this shortcoming, a novel transport assay based on solid-supported membrane-based electrophysiology (SSME) is presented. Key findings of the present SSME studies include the first recordings of electrophysiological properties, a pH dependence analysis, an assessment of PHT1 substrate selectivity, as well as the transport kinetics of the identified substrates. In contrast to previous work, PHT1 is studied in its native lysosomal environment. Moreover, observed substrate selectivity is validated by molecular docking. Overall, this new SSME-based assay is expected to contribute to unlocking the pharmacological potential of PHT1 and to deepen the understanding of its functional properties.

Structure-based reshaping of a new ketoreductase from Sphingobacterium siyangense SY1 toward α-haloacetophenones.

Ketoreductases play an indispensable role in the asymmetric synthesis of chiral drug intermediates, and an in-depth understanding of their substrate selectivity can improve the efficiency of enzyme engineering. In this endeavor, a new short-chain dehydrogenase/reductase (SDR) SsSDR1 identified from Sphingobacterium siyangense SY1 by gene mining method was successfully cloned and functionally expressed in Escherichia coli. Its activity against halogenated acetophenones has been tested and the results illustrated that SsSDR1-WT exhibits high activity for 3,5-bis(trifluoromethyl)acetophenone (1f), an important precursor in the synthesis of aprepitant. In addition, SsSDR1-WT showed obvious substrate preference for acetophenones without α-halogen substitution compared to their α-halogen analogs. To explore the structural basis of substrate selectivity, the X-ray crystal structures of SsSDR1-WT in its apo form and the complex structure with NAD were resolved. Taking 2-chloro-1-(3, 4-difluorophenyl) ethanone (1i) as the representative α-haloacetophenone, the key sites affecting substrate selectivity of SsSDR1-WT were identified and through the rational remodeling of the cavities C1 and C2 of SsSDR1, an excellent mutant I144A/S153L with significantly improved activity against α-halogenated acetophenones was obtained. The asymmetric catalysis of 1f and 1i was performed at the scale of 50 mL, and the space-time yields (STY) of the two were 1200 and 6000 g/L∙d, respectively. This study not only provides valuable biocatalysts for halogenated acetophenones, but also yields insights into the relationship between the substrate-binding pocket and substrate selectivity.

Fidelity in plant hormone modifications catalyzed by Arabidopsis GH3 acyl acid amido synthetases.

GRETCHEN HAGEN 3 (GH3) acyl acid amido synthetases conjugate amino acids to acyl acid hormones to either activate or inactivate the hormone molecule. The largest subgroup of GH3 proteins modify the growth-promoting hormone auxin (indole-3-acetic acid; IAA) with the second largest class activating the defense hormone jasmonic acid (JA). The two-step reaction mechanism of GH3 proteins provides a potential proofreading mechanism to ensure fidelity of hormone modification. Examining pyrophosphate release in the first-half reaction of Arabidopsis GH3 proteins that modify IAA (AtGH3.2/YDK2, AtGH3.5/WES1, AtGH3.17/VAS2), JA (AtGH3.11/JAR1), and other acyl acids (AtGH3.7, AtGH3.12/PBS3) indicates that acyl acid-AMP intermediates are hydrolyzed into acyl acid and AMP in the absence of the amino acid, a typical feature of pre-transfer editing mechanisms. Single-turnover kinetic analysis of AtGH3.2/YDK2 and AtGH3.5/WES1 shows that non-cognate acyl acid-adenylate intermediates are more rapidly hydrolyzed than the cognate IAA-adenylate. In contrast, AtGH3.11/JAR1 only adenylates JA, not IAA. While some of the auxin-conjugating GH3 proteins in Arabidopsis (i.e., AtGH3.5/WES1) accept multiple acyl acid substrates, others, like AtGH3.2/YDK2, are specific for IAA; however, both these proteins share similar active site residues. Biochemical analysis of chimeric variants of AtGH3.2/YDK2 and AtGH3.5/WES1 indicates that the C-terminal domain contributes to selection of cognate acyl acid substrates. These findings suggest that the hydrolysis of non-cognate acyl acid-adenylate intermediates, or proofreading, proceeds via a slowed structural switch that provides a checkpoint for fidelity before the full reaction proceeds.

Maximizing the potential of nitrilase: Unveiling their diversity, catalytic proficiency, and versatile applications.

Nitrilases represent a distinct class of enzymes that play a pivotal role in catalyzing the hydrolysis of nitrile compounds, leading to the formation of corresponding carboxylic acids. These enzymatic entities have garnered significant attention across a spectrum of industries, encompassing pharmaceuticals, agrochemicals, and fine chemicals. Moreover, their significance has been accentuated by mounting environmental pressures, propelling them into the forefront of biodegradation and bioremediation endeavors. Nevertheless, the natural nitrilases exhibit intrinsic limitations such as low thermal stability, narrow substrate selectivity, and inadaptability to varying environmental conditions. In the past decade, substantial efforts have been made in elucidating the structural underpinnings and catalytic mechanisms of nitrilase, providing basis for engineering of nitrilases. Significant breakthroughs have been made in the regulation of nitrilases with ideal catalytic properties and application of the enzymes for industrial productions. This review endeavors to provide a comprehensive discourse and summary of recent research advancements related to nitrilases, with a particular emphasis on the elucidation of the structural attributes, catalytic mechanisms, catalytic characteristics, and strategies for improving catalytic performance of nitrilases. Moreover, the exploration extends to the domain of process engineering and the multifarious applications of nitrilases. Furthermore, the future development trend of nitrilases is prospected, providing important guidance for research and application in the related fields.

Effect of pinoresinol-lariciresinol reductases on biosynthesis of lignans with substrate selectivity in Schisandra chinensis.

Schisandra lignans are the main bioactive compounds found in Schisandra chinensis fruits, such as schisandrol lignans and schisandrin lignans, which play important roles in organ protection or other clinical roles. Pinoresinol-lariciresinol reductase (PLR) plays a pivotal role in plant lignan biosynthesis, however, limited research has been conducted on S. chinensis PLR to date. This study identified five genes as ScPLR, successfully cloned their coding sequences, and elucidated their catalytic capabilities. ScPLR3-5 could recognize both pinoresinol and lariciresinol as substrates, and convert them into lariciresinol and secoisolariciresinol, respectively, while ScPLR2 exclusively catalyzed the conversion of (+)-pinoresinol into (+)-lariciresinol. Transcript-metabolite correlation analysis indicated that ScPLR2 exhibited unique properties that differed from the other members. Molecular docking and site-directed mutagenesis revealed that Phe271 and Leu40 in the substrate binding motif were crucial for the catalytic activity of ScPLR2. This study serves as a foundation for understanding the essential enzymes involved in schisandra lignan biosynthesis.

Endomembrane-biased dimerization of ABCG16 and ABCG25 transporters determines their substrate selectivity in ABA-regulated plant growth and stress responses.

ATP-binding cassette (ABC) transporters are integral membrane proteins that have evolved diverse functions fulfilled via the transport of various substrates. In Arabidopsis, the G subfamily of ABC proteins is particularly abundant and participates in multiple signaling pathways during plant development and stress responses. In this study, we revealed that two Arabidopsis ABCG transporters, ABCG16 and ABCG25, engage in ABA-mediated stress responses and early plant growth through endomembrane-specific dimerization-coupled transport of ABA and ABA-glucosyl ester (ABA-GE), respectively. We first revealed that ABCG16 contributes to osmotic stress tolerance via ABA signaling. More specifically, ABCG16 induces cellular ABA efflux in both yeast and plant cells. Using FRET analysis, we showed that ABCG16 forms obligatory homodimers for ABA export activity and that the plasma membrane-resident ABCG16 homodimers specifically respond to ABA, undergoing notable conformational changes. Furthermore, we demonstrated that ABCG16 heterodimerizes with ABCG25 at the endoplasmic reticulum (ER) membrane and facilitates the ER entry of ABA-GE in both Arabidopsis and tobacco cells. The specific responsiveness of the ABCG16-ABCG25 heterodimer to ABA-GE and the superior growth of their double mutant support an inhibitory role of these two ABCGs in early seedling establishment via regulation of ABA-GE translocation across the ER membrane. Our endomembrane-specific analysis of the FRET signals derived from the homo- or heterodimerized ABCG complexes allowed us to link endomembrane-biased dimerization to the translocation of distinct substrates by ABCG transporters, providing a prototypic framework for understanding the omnipotence of ABCG transporters in plant development and stress responses.

Structural determinants for tRNA selective cleavage by RNase 2/EDN.

tRNA-derived fragments (tRFs) have emerged as key players of immunoregulation. Some RNase A superfamily members participate in the shaping of the tRFs population. By comparing wild-type and knockout macrophage cell lines, our previous work revealed that RNase 2 can selectively cleave tRNAs. Here, we confirm the in vitro protein cleavage pattern by screening of synthetic tRNAs, single-mutant variants, and anticodon-loop DNA/RNA hairpins. By sequencing of tRF products, we identified the cleavage selectivity of recombinant RNase 2 with base specificity at B1 (U/C) and B2 (A) sites, consistent with a previous cellular study. Lastly, protein-hairpin complexes were predicted by MD simulations. Results reveal the contribution of the α1, loop 3 and loop 4, and ß6 RNase 2 regions, where residues Arg36/Asn39/Gln40/Asn65/Arg68/Arg132 provide interactions, spanning from P-1 to P2 sites that are essential for anticodon loop recognition. Knowledge of RNase 2-specific tRFs generation might guide new therapeutic approaches for infectious and immune-related diseases.

Substrate selectivity and unique sequence signatures in SWEET/semiSWEET homologs of four taxonomic groups: Sequence analysis and phylogenetic studies.

The recently discovered SWEET (Sugar Will Eventually be Exported Transporter) proteins are involved in the selective transport of monosaccharides and disaccharides. The prokaryotic counterparts, semiSWEETs, form dimers with each monomer forming a triple-helix transmembrane bundle (THB). The longer eukaryotic SWEETs have seven transmembrane helices with two THBs and a linker helix. Structures of semiSWEETs/SWEETs have been determined experimentally. Experimental studies revealed the role of plant SWEETs in vital physiological processes and identified residues responsible for substrate selectivity. However, SWEETs/semiSWEETs from metazoans and bacteria are not characterized. In this study, we used structure-based sequence alignment and compared more than 2000 SWEET/semiSWEETs from four different taxonomic groups. Conservation of residue/chemical property was examined at all positions. Properties of clades/subclades of phylogenetic trees from each taxonomic group were analyzed. Conservation pattern of known residues in the selectivity-filter was used to predict the substrate preference of plant SWEETs and some clusters of metazoans and bacteria. Some residues at the gating and substrate-binding regions, pore-facing positions and at the helix-helix interface are conserved across all taxonomic groups. Conservation of polar/charged residues at specific pore-facing positions, helix-helix interface and in loops seems to be unique for plant SWEETs. Overall, the number of conserved residues is less in metazoan SWEETs. Plant and metazoan SWEETs exhibit high conservation of four and three proline residues respectively in "proline tetrad." Further experimental studies can validate the predicted substrate selectivity and significance of conserved polar/charged/aromatic residues at structurally and functionally important positions of SWEETs/semiSWEETs in plants, metazoans and bacteria.

Reconstitution of RNA cap methylation reveals different features of SARS-CoV-2 and SARS-CoV methyltransferases.

Cap RNA methylations play important roles in the replication, evasion of host RNA sensor recognition, and pathogenesis. Coronaviruses possess both guanine N7- and 2'-O-ribose methyltransferases (N7-MTase and 2'-O-MTase) encoded by nonstructural protein (nsp) 14 and nsp16/10 complex, respectively. In this study, we reconstituted the two-step RNA methylations of N7-MTase and 2'-O-MTase of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro and demonstrated its common and different features in comparison with that of SARS-CoV. We revealed that the nsp16/10 2'-O-MTase of SARS-CoV-2 has a broader substrate selectivity than the counterpart of SARS-CoV and can accommodate both unmethylated and uncapped RNA substrates in a sequence-independent manner. Most intriguingly, the substrate selectivity of nsp16/10 complex is not determined by the apoenzyme of nsp16 MTase but by its cofactor nsp10. These results provide insight into the unique features of SARS-CoV-2 MTases and may help develop strategies to precisely intervene in the methylation pathway and pathogenesis of SARS-CoV-2.