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.

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.

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.

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.

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.

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.

Development of a Highly Selective NAD+ -Dependent Glyceraldehyde Dehydrogenase and its Application in Minimal Cell-Free Enzyme Cascades.

Anthropogenic climate change has been caused by over-exploitation of fossil fuels and CO2 emissions. To counteract this, the chemical industry has shifted its focus to sustainable chemical production and the valorization of renewable resources. However, the biggest challenges in biomanufacturing are technical efficiency and profitability. In our minimal cell-free enzyme cascade generating pyruvate as the central intermediate, the NAD+ -dependent, selective oxidation of D-glyceraldehyde was identified as a key reaction step to improve the overall cascade flux. Successive genome mining identified one candidate enzyme with 24-fold enhanced activity and another whose stability is unaffected in 10 % (v/v) ethanol, the final product of our model cascade. Semi-rational engineering improved the substrate selectivity of the enzyme up to 21-fold, thus minimizing side reactions in the one-pot enzyme cascade. The final biotransformation of D-glucose showed a continuous linear production of ethanol (via pyruvate) to a final titer of 4.9 % (v/v) with a molar product yield of 98.7 %. Due to the central role of pyruvate in diverse biotransformations, the optimized production module has great potential for broad biomanufacturing applications.

Substrate Selectivity Imparted by Self-Assembled Molecular Containers and Catalysts.

Recent trends in catalysis are devoted to mimicking some peculiar features of enzymes like site selectivity, through functional group recognition, and substrate selectivity, through recognition of the entire surface of the substrate. The latter is a specific feature of enzymes that is seldomly present in homogeneous catalysis. Supramolecular catalysis, thanks to the self-assembly of simple subunits, enables the creation of cavities and surfaces whose confinement effects drive the preferential binding of a substrate among others with consequent substrate selectivity. The topic is an emerging field that exploits recognition phenomena to discriminate the reagents based on their size and shape. This review deals this cutting-edge field of research covering examples of supramolecular self-assembled molecular containers and catalysts operating in organic as well as aqueous media, with special emphasis for catalytic systems dealing with direct competitive experiments involving two or more substrates.

The S1'-S3' Pocket of the SARS-CoV-2 Main Protease Is Critical for Substrate Selectivity and Can Be Targeted with Covalent Inhibitors.

The main protease (Mpro ) of SARS-CoV-2 is a well-characterized target for antiviral drug discovery. To date, most antiviral drug discovery efforts have focused on the S4-S1' pocket of Mpro ; however, it is still unclear whether the S1'-S3' pocket per se can serve as a new site for drug discovery. In this study, the S1'-S3' pocket of Mpro was found to differentially recognize viral peptidyl substrates. For instance, S3' in Mpro strongly favors Phe or Trp, and S1' favors Ala. The peptidyl inhibitor D-4-77, which possesses an α-bromoacetamide warhead, was discovered to be a promising inhibitor of Mpro , with an IC50 of 0.95 µM and an antiviral EC50 of 0.49 µM. The Mpro /inhibitor co-crystal structure confirmed the binding mode of the inhibitor to the S1'-S3' pocket and revealed a covalent mechanism. In addition, D-4-77 functions as an immune protectant and suppresses SARS-CoV-2 Mpro -induced antagonism of the host NF-κB innate immune response. These findings indicate that the S1'-S3' pocket of SARS-CoV-2 Mpro is druggable, and that inhibiting SARS-CoV-2 Mpro can simultaneously protect human innate immunity and inhibit virion assembly.

Functions and substrate selectivity of diacylglycerol acyltransferases from Mortierella alpina.

Mortierella alpina produces various polyunsaturated fatty acids in the form of triacylglycerols (TAG). Diacylglycerol acyltransferase (DGAT) catalyzes the binding of acyl-CoA to diacylglycerol to form TAG and is the key enzyme involved in TAG synthesis. A variety of DGATs are present in M. alpina; however, comparative analysis of the functional properties and substrate selectivity of these DGATs is insufficient. In this study, DGAT1 (MaDGAT1A/1B/1C) and DGAT2 (MaDGAT2A/2B) isoforms from M. alpina were analyzed and heterologously expressed in S. cerevisiae H1246. The results showed that MaDGAT1A/1B/2A/2B were able to restore TAG synthesis, and the corresponding TAG content in recombinant yeasts was 2.92 ± 0.42%, 3.62 ± 0.22%, 0.86 ± 0.34%, and 0.18 ± 0.09%, respectively. In S. cerevisiae H1246, MaDGAT1A preferred C16:1 among monounsaturated fatty acids, MaDGAT1B preferred C16:0 among saturated fatty acids (SFAs), and MaDGAT2A/2B preferred C18:0 among SFAs. Under exogenous addition of polyunsaturated fatty acids (PUFAs), MaDGAT1A and 2A preferentially assembled linoleic acid into TAG, and MaDGAT2B had substrate selectivity for eicosapentaenoic and linoleic acids in ω-6 PUFAs. In vitro, MaDGAT1A showed no obvious acyl-CoA selectivity and MaDGAT1B preferred C20:5-CoA. MaDGAT1A/1B preferred C18:1/C18:1-DAG compared with C20:4/C20:4-DAG. This study indicates that MaDGATs have the potential to be used in the production of LA/EPA-rich TAG and provide a reference for improving the production of TAGs in oleaginous fungi. KEY POINTS: • MaDGAT1A preferred C16:1 among MUFAs, MaDGAT1B and MaDGAT2A/2B preferred C16:0 and C18:0 among SFAs, respectively • MaDGAT1A/2A preferentially assembled linoleic acid into TAG, and MaDGAT2B has substrate selectivity for eicosapentaenoic acid and linoleic acid in ω-6 PUFAs • MaDGAT1A showed no obvious acyl-CoA selectivity, and MaDGAT1B preferred C20:5-CoA. MaDGAT1A/1B preferred to select C18:1/C18:1-DAG compared with C20:4/C20:4-DAG.

Crystal structures and kinetic studies of a laboratory evolved aldehyde reductase explain the dramatic shift of its new substrate specificity.

The Fe2+-dependent E. coli enzyme FucO catalyzes the reversible interconversion of short-chain (S)-lactaldehyde and (S)-1,2-propanediol, using NADH and NAD+ as cofactors, respectively. Laboratory-directed evolution experiments have been carried out previously using phenylacetaldehyde as the substrate for screening catalytic activity with bulky substrates, which are very poorly reduced by wild-type FucO. These experiments identified the N151G/L259V double mutant (dubbed DA1472) as the most active variant with this substrate via a two-step evolutionary pathway, in which each step consisted of one point mutation. Here the crystal structures of DA1472 and its parent D93 (L259V) are reported, showing that these amino acid substitutions provide more space in the active site, though they do not cause changes in the main-chain conformation. The catalytic activity of DA1472 with the physiological substrate (S)-lactaldehyde and a series of substituted phenylacetaldehyde derivatives were systematically quantified and compared with that of wild-type as well as with the corresponding point-mutation variants (N151G and L259V). There is a 9000-fold increase in activity, when expressed as kcat/KM values, for DA1472 compared with wild-type FucO for the phenylacetaldehyde substrate. The crystal structure of DA1472 complexed with a non-reactive analog of this substrate (3,4-dimethoxyphenylacetamide) suggests the mode of binding of the bulky group of the new substrate. These combined structure-function studies therefore explain the dramatic increase in catalytic activity of the DA1472 variant for bulky aldehyde substrates. The structure comparisons also suggest why the active site in which Fe2+ is replaced by Zn2+ is not able to support catalysis.

RBM33 is a unique m6A RNA-binding protein that regulates ALKBH5 demethylase activity and substrate selectivity.

Regulation of RNA substrate selectivity of m6A demethylase ALKBH5 remains elusive. Here, we identify RNA-binding motif protein 33 (RBM33) as a previously unrecognized m6A-binding protein that plays a critical role in ALKBH5-mediated mRNA m6A demethylation of a subset of mRNA transcripts by forming a complex with ALKBH5. RBM33 recruits ALKBH5 to its m6A-marked substrate and activates ALKBH5 demethylase activity through the removal of its SUMOylation. We further demonstrate that RBM33 is critical for the tumorigenesis of head-neck squamous cell carcinoma (HNSCC). RBM33 promotes autophagy by recruiting ALKBH5 to demethylate and stabilize DDIT4 mRNA, which is responsible for the oncogenic function of RBM33 in HNSCC cells. Altogether, our study uncovers the mechanism of selectively demethylate m6A methylation of a subset of transcripts during tumorigenesis that may explain demethylation selectivity in other cellular processes, and we showed its importance in the maintenance of tumorigenesis of HNSCC.

Structure-Based Redesign of a Methanol Oxidase into an "Aryl Alcohol Oxidase" for Enzymatic Synthesis of Aromatic Flavor Compounds.

Alcohol oxidases (AOxs) catalyze the aerobic oxidation of alcohols to the corresponding carbonyl products (aldehydes or ketones), producing only H2O2 as the byproduct. The majority of known AOxs, however, have a strong preference for small, primary alcohols, limiting their broad applicability, e.g., in the food industry. To broaden the product scope of AOxs, we performed structure-guided enzyme engineering of a methanol oxidase from Phanerochaete chrysosporium (PcAOx). The substrate preference was extended from methanol to a broad range of benzylic alcohols by modifying the substrate binding pocket. A mutant (PcAOx-EFMH) with four substitutions exhibited improved catalytic activity toward benzyl alcohols with increased conversion and kcat toward the benzyl alcohol from 11.3 to 88.9% and from 0.5 to 2.6 s-1, respectively. The molecular basis for the change of substrate selectivity was analyzed by molecular simulation.

Identification of nonhistone substrates of the lysine methyltransferase PRDM9.

Lysine methylation is a dynamic, posttranslational mark that regulates the function of histone and nonhistone proteins. Many of the enzymes that mediate lysine methylation, known as lysine methyltransferases (KMTs), were originally identified to modify histone proteins but have also been discovered to methylate nonhistone proteins. In this work, we investigate the substrate selectivity of the KMT PRDM9 to identify both potential histone and nonhistone substrates. Though normally expressed in germ cells, PRDM9 is significantly upregulated across many cancer types. The methyltransferase activity of PRDM9 is essential for double-strand break formation during meiotic recombination. PRDM9 has been reported to methylate histone H3 at lysine residues 4 and 36; however, PRDM9 KMT activity had not previously been evaluated on nonhistone proteins. Using lysine-oriented peptide libraries to screen potential substrates of PRDM9, we determined that PRDM9 preferentially methylates peptide sequences not found in any histone protein. We confirmed PRDM9 selectivity through in vitro KMT reactions using peptides with substitutions at critical positions. A multisite λ-dynamics computational analysis provided a structural rationale for the observed PRDM9 selectivity. The substrate selectivity profile was then used to identify putative nonhistone substrates, which were tested by peptide spot array, and a subset was further validated at the protein level by in vitro KMT assays on recombinant proteins. Finally, one of the nonhistone substrates, CTNNBL1, was found to be methylated by PRDM9 in cells.

Chemodiversity of soil organic matters determines biodegradation of polychlorinated biphenyls by a graphene oxide-assisted bacterial agent.

A promising strategy for degrading persistent organic pollutants (POPs) in soil is amendment with nanomaterial-assisted functional bacteria. However, the influence of soil organic matter chemodiversity on the performance of nanomaterial-assisted bacterial agents remains unclear. Herein, different types of soil (Mollisol soil, MS; Ultisol soil, US; and Inceptisol soil, IS) were inoculated with a graphene oxide (GO)-assisted bacterial agent (Bradyrhizobium diazoefficiens USDA 110, B. diazoefficiens USDA 110) to investigate the association between soil organic matter chemodiversity and stimulation of polychlorinated biphenyl (PCB) degradation. Results indicated that the high-aromatic solid organic matter (SOM) inhibited PCB bioavailability, and lignin-dominant dissolved organic matter (DOM) with high biotransformation potential was a favored substrate for all PCB degraders, which led to no stimulation of PCB degradation in MS. Differently, high-aliphatic SOM in US and IS promoted PCB bioavailability. The high/low biotransformation potential of multiple DOM components (e.g., lignin, condensed hydrocarbon, unsaturated hydrocarbon, etc.) in US/IS further resulted to the enhanced PCB degradation by B. diazoefficiens USDA 110 (up to 30.34%) /all PCB degraders (up to 17.65%), respectively. Overall, the category and biotransformation potential of DOM components and the aromaticity of SOM collaboratively determine the stimulation of GO-assisted bacterial agent on PCB degradation.

Molecular Insights into Bifunctional Ambruticin DH3 for Substrate Specificity and Catalytic Mechanism.

Dehydratase (DH), a domain located at polyketide synthase (PKS) modules, commonly catalyzes the dehydration of ß-hydroxy to an α,ß-unsaturated acyl intermediate. As a unique bifunctional dehydratase, AmbDH3 (the DH domain of module 3 of the ambruticin PKS) is verified to be responsible for both dehydration and the following pyran-forming cyclization. Besides, in vitro studies showed that its catalytic efficiency varies with different chiral substrates. However, the detailed molecular mechanism of AmbDH3 remains unclear. In this work, the structural rationale for the substrate specificity (2R/2S- and 6R/6S-substrates) in AmbDH3 was elucidated and the complete reaction pathways including dehydration and cyclization were presented. Both MD simulations and binding free energy calculations indicated AmbDH3 had a stronger preference for 2R-substrates (2R6R-2, 2R6S-3) than 2S-substrates (2S6R-1), and residue H51 and G61 around the catalytic pocket were emphasized by forming stable hydrogen bonds with 2R-substrates. In addition, AmbDH3's mild tolerance at C6 was explained by comparison of substrate conformation and hydrogen bond network in 6S- and 6R-substrate systems. The QM/MM results supported a consecutive one-base dehydration and cyclization mechanism for 2R6S-3 substrate with the energy barrier of 25.2 kcal mol-1 and 24.5 kcal mol-1 , respectively. Our computational results uncover the substrate recognition and catalytic process of the first bifunctional dehydratase-cyclase AmbDH3, which will shed light on the application of multifunctional DH domains in PKSs for diverse natural product analogs and benefit the chemoenzymatic synthesis of stereoselective pyran-containing products.