Superfamily Of Enzymes Research Articles

Biological Characteristics of the Cytochrome P 450 Family and the Mechanism of Terpinolene Metabolism in Hyalomma asiaticum (Acari: Ixodidae)

The CYP450 enzyme is a superfamily enzyme ubiquitously found in nearly all organisms, playing a vital role in the metabolism of both endogenous and exogenous compounds, and in biosynthesis. Unfortunately, an understanding of its classification, functions, expression characteristics, and other biological traits in Hyalomma asiaticum, a vector for Crimean–Congo Hemorrhagic Fever, as well as of the genes implicated in its natural product metabolism, is lacking. Towards this end, this study has identified 120 H. asiaticum CYP450 genes via transcriptome data in the face of a joint genome threat from terpinolene. The proteins these genes encode are of higher molecular weight, devoid of a signal peptide, and composed of unstable hydrophobic proteins principally containing 1–3 variable transmembrane regions. Phylogenetic evolution classifies these H. asiaticum CYP450 genes into four subfamilies. These genes all encompass complete CYP450 conserved domains, and five specific conserved motifs, albeit with different expression levels. GO and KEGG annotation findings suggest a widespread distribution of these CYP450 genes in many physiological systems, predominantly facilitating lipid metabolism, terpenoid compound metabolism, and polyketone compound metabolism, as well as cofactor and vitamin metabolism at a cellular level. Molecular docking results reveal a hydrophobic interaction between the ARG-103, ARG-104, LEU-106, PHE-109, and ILE-119 amino acid residues in CYP3A8, which is primarily expressed in the fat body, and terpinolene, with a notably up-regulated expression, with affinity = −5.6 kcal/mol. The conservation of these five key amino acid residues varies across 12 tick species, implying differences in terpinolene metabolism efficacy among various tick species. This study thereby fills an existing knowledge gap regarding the biological characteristics of H. asiaticum CYP450 genes and paves the way for further research into the functions of these particular genes.

CG17192 is a Phospholipase That Regulates Signaling Lipids in the Drosophila Gut upon Infection.

The chemoproteomics technique, activity-based protein profiling (ABPP), has proven to be an invaluable tool in assigning functions to enzymes. The serine hydrolase (SH) enzyme superfamily, in particular, has served as an excellent example in displaying the versatility of various ABPP platforms and has resulted in a comprehensive cataloging of the biochemical activities associated within this superfamily. Besides SHs, in mammals, several other enzyme classes have been thoroughly investigated using ABPP platforms. However, the utility of ABPP platforms in fly models remains underexplored. Realizing this knowledge gap, leveraging complementary ABPP platforms, we reported the full array of SH activities during various developmental stages and adult tissues in the fruit fly (Drosophila melanogaster). Following up on this study, using ABPP, we mapped SH activities in adult fruit flies in an infection model and found that a gut-resident lipase CG17192 showed increased activity during infection. To assign a biological function to this uncharacterized lipase, we performed an untargeted lipidomics analysis and found that phosphatidylinositols were significantly elevated when CG17192 was depleted in the adult fruit fly gut. Next, we overexpressed this lipase in insect cells, and using biochemical assays, we show that CG17192 is a secreted enzyme that has phospholipase C (PLC) type activity, with phosphatidylinositol being a preferred substrate. Finally, we show during infection that heightened CG17192 regulates phosphatidylinositol levels and, by doing so, likely modulates signaling pathways in the adult fruit fly gut that might be involved in the resolution of this pathophysiological condition.

Fe(II) and 2-oxoglutarate-dependent dioxygenases for natural product synthesis: molecular insights into reaction diversity.

Covering: up to 2024Fe(II) and 2-oxoglutarate-dependent dioxygenases (Fe/2OG DOs) are a superfamily of enzymes that play important roles in a variety of catalytic reactions, including hydroxylation, ring formation, ring reconstruction, desaturation, and demethylation. Each member of this family has similarities in their overall structure, but they have varying specific differences, making Fe/2OG DOs attractive for catalytic diversity. With the advancement of current research, more Fe/2OG DOs have been discovered, and their catalytic scope has been further broadened; however, apart from hydroxylation, many reaction mechanisms have not been accurately demonstrated, and there is a lack of a systematic understanding of their molecular basis. Recently, an increasing number of X-ray structures of Fe/2OG DOs have provided new insights into the structural basis of their function and substrate-binding properties. This structural information is essential for understanding catalytic mechanisms and mining potential catalytic reactions. In this review, we summarize most of the Fe/2OG DOs whose structures have been resolved in recent years, focus on their structural features, and explore the relationships between various structural elements and unique catalytic mechanisms and their associated reaction type classification.

Computational Stabilization of a Non‐heme Iron Enzyme Enables Efficient Evolution of New Function

Deep learning tools for enzyme design are rapidly emerging, and there is a critical need to evaluate their effectiveness in engineering workflows. Here we show that the deep learning‐based tool ProteinMPNN can be used to redesign Fe(II)/αKG superfamily enzymes for greater stability, solubility, and expression while retaining both native activity and industrially‐relevant non‐native functions. This superfamily has diverse catalytic functions and could provide a rich new source of biocatalysts for synthesis and industrial processes. Through systematic comparisons of directed evolution trajectories for a non‐native, remote C(sp3)‐H hydroxylation reaction, we demonstrate that the stabilized redesign can be evolved more efficiently than the wild‐type enzyme. After three rounds of directed evolution, we obtained a 6‐fold activity increase from the wild‐type parent and an 80‐fold increase from the stabilized variant. To generate the initial stabilized variant, we identified multiple structural and sequence constraints to preserve catalytic function. We applied these criteria to produce stabilized, catalytically active variants of a second Fe(II)/αKG enzyme, suggesting that the approach is generalizable to additional members of the Fe(II)/αKG superfamily. ProteinMPNN is user‐friendly and widely‐accessible, and our results provide a framework for the routine implementation of deep learning‐based protein stabilization tools in directed evolution workflows for novel biocatalysts.

Computational Stabilization of a Non-heme Iron Enzyme Enables Efficient Evolution of New Function.

Deep learning tools for enzyme design are rapidly emerging, and there is a critical need to evaluate their effectiveness in engineering workflows. Here we show that the deep learning-based tool ProteinMPNN can be used to redesign Fe(II)/αKG superfamily enzymes for greater stability, solubility, and expression while retaining both native activity and industrially-relevant non-native functions. This superfamily has diverse catalytic functions and could provide a rich new source of biocatalysts for synthesis and industrial processes. Through systematic comparisons of directed evolution trajectories for a non-native, remote C(sp3)-H hydroxylation reaction, we demonstrate that the stabilized redesign can be evolved more efficiently than the wild-type enzyme. After three rounds of directed evolution, we obtained a 6-fold activity increase from the wild-type parent and an 80-fold increase from the stabilized variant. To generate the initial stabilized variant, we identified multiple structural and sequence constraints to preserve catalytic function. We applied these criteria to produce stabilized, catalytically active variants of a second Fe(II)/αKG enzyme, suggesting that the approach is generalizable to additional members of the Fe(II)/αKG superfamily. ProteinMPNN is user-friendly and widely-accessible, and our results provide a framework for the routine implementation of deep learning-based protein stabilization tools in directed evolution workflows for novel biocatalysts.

A single Leishmania adenylate–forming enzyme of the ANL superfamily generates both acetyl- and acetoacetyl-CoA

Leishmania, a protozoan parasite, is responsible for significant morbidity and mortality worldwide, manifesting as cutaneous, mucocutaneous, and visceral leishmaniasis. These diseases pose a substantial burden, especially in impoverished regions with limited access to effective medical treatments. Current therapies are toxic, have low efficacy, and face growing resistance. Understanding the metabolic pathways of Leishmania, particularly those differing from its host, can unveil potential therapeutic targets. In this study, we investigated the acetyl-CoA synthetase (ACS) enzyme from Leishmania infantum (LiAcs1), which, unlike many organisms, also exhibits acetoacetyl-CoA synthetase (KBC) activity. This dual functionality is unique among ANL superfamily enzymes and crucial for the parasite's reliance on leucine catabolism, energy production and sterol biosynthesis. Our biochemical characterization of LiAcs1 revealed its ability to utilize both acetate and acetoacetate substrates. Additionally, LiAcs1 displayed a distinct CoA substrate inhibition pattern, partially alleviated by acetoacetate. Structural analysis provided insights into the substrate binding flexibility of LiAcs1, highlighting a more promiscuous substrate pocket compared to other ACS or KBC-specific enzymes. Substrate mimetics elucidated its ability to accommodate both small and large AMP-ester derivatives, contributing to its dual ACS/KBC functionality. These findings not only advance our understanding of Leishmania metabolism but also present LiAcs1 as a promising drug target. The dual functionality of LiAcs1 underscores the potential for developing selective inhibitors that could disrupt critical metabolic pathways across Leishmania spp. as it appears this enzyme is highly conserved across this genus. This paves the way for developing novel effective treatments against this devastating disease.

Biochemical and structural insights into a 5’ to 3’ RNA ligase reveal a potential role in tRNA ligation

ATP-grasp superfamily enzymes contain a hand-like ATP-binding fold and catalyze a variety of reactions using a similar catalytic mechanism. More than 30 protein families are categorized in this superfamily, and they are involved in a plethora of cellular processes and human diseases. Here, we identify C12orf29 (RLIG1) as an atypical ATP-grasp enzyme that ligates RNA. Human RLIG1 and its homologs autoadenylate on an active site Lys residue as part of a reaction intermediate that specifically ligates RNA halves containing a 5'-phosphate and a 3'-hydroxyl. RLIG1 binds tRNA in cells and can ligate tRNA within the anticodon loop in vitro. Transcriptomic analyses of Rlig1 knockout mice revealed significant alterations in global tRNA levels in the brains of female mice, but not in those of male mice. Furthermore, crystal structures of a RLIG1 homolog from Yasminevirus bound to nucleotides revealed a minimal and atypical RNA ligase fold with a conserved active site architecture that participates in catalysis. Collectively, our results identify RLIG1 as an RNA ligase and suggest its involvement in tRNA biology.

Not all cytochrome b5s are created equal: how a specific CytB5 boosts forskolin biosynthesis in Saccharomyces cerevisiae

Cytochrome B5s, or CytB5s, are small heme-binding proteins, ubiquitous across all kingdoms of life that serve mainly as electron donors to enzymes engaged in oxidative reactions. They often function as redox partners of the cytochrome P450s (CYPs), a superfamily of enzymes participating in multiple biochemical processes. In plants, CYPs catalyze key reactions in the biosynthesis of plant specialized metabolites with their activity dependent on electron donation often from cytochrome P450 oxidoreductases (CPRs or PORs). In eukaryotic microsomal CYPs, CytB5s frequently participate in the electron transfer process although their exact role remains understudied, especially in plant systems. In this study, we assess the role of CytB5s in the heterologous biotechnological production of plant specialized metabolites in yeast. For this, we used as a case-study the biosynthesis of forskolin - a bioactive diterpenoid produced exclusively from the plant Coleus forskohlii. The complete biosynthetic pathway for forskolin is known and includes three CYP enzymes. We reconstructed the entire forskolin pathway in the yeast Saccharomyces cerevisiae, and upon co-expression of the three CytB5s - identified in C. forskohlii transcriptomes - alleviation of a CYP-related bottleneck step was noticed only when a specific CytB5, CfCytB5A, was used. Co-expression of CfCytB5A in yeast, in combination with forskolin pathway engineering, resulted in forskolin production at titers of 1.81 g/L in a bioreactor. Our findings demonstrate that CytB5s not only play an important role in plant specialized metabolism but also, they can interact with precision with specific CYPs, indicating that the properties of CytB5s are far from understood. Moreover, our work highlights how CytB5s may act as indispensable components in the sustainable microbial production of plant metabolites, when their biosynthetic pathways involve CYP enzymes.

Cytochromes P450 evolution in the plant terrestrialization context.

Plants started to colonize land around 500 million years ago. It meant dealing with new challenges like absence of buoyancy, water and nutrients shortage, increased light radiation, reproduction on land, and interaction with new microorganisms. This obviously required the acquisition of novel functions and metabolic capacities. Cytochrome P450 (CYP) monooxygenases form the largest superfamily of enzymes and are present to catalyse critical and rate-limiting steps in most plant-specific pathways. The different families of CYP enzymes are typically associated with specific functions. CYP family emergence and evolution in the green lineage thus offer the opportunity to obtain a glimpse into the timing of the evolution of the critical functions that were required (or became dispensable) for the plant transition to land. Based on the analysis of currently available genomic data, this review provides an evolutionary history of plant CYPs in the context of plant terrestrialization and describes the associated functions in the different lineages. Without surprise it highlights the relevance of the biosynthesis of antioxidants and UV screens, biopolymers, and critical signalling pathways. It also points to important unsolved questions that would deserve to be answered to improve our understanding of plant adaptation to challenging environments and the management of agricultural traits. This article is part of the theme issue 'The evolution of plant metabolism'.

Phosphodiesterases: Evolving Concepts and Implications for Human Therapeutics.

Phosphodiesterases (PDEs) are a superfamily of enzymes that hydrolyze cyclic nucleotides. While the 11 PDE subfamilies share common features, key differences confer signaling specificity. The differences include substrate selectivity, enzymatic activity regulation, tissue expression, and subcellular localization. Selective inhibitors of each subfamily have elucidated the protean role of PDEs on normal cell function. PDEs are also linked to diseases, some of which affect the immune, cardiac, and vascular systems. Selective PDE inhibitors are clinically used to treat these specific disorders. Ongoing preclinical studies and clinical trials are likely to lead to the approval of additional PDE-targeting drugs for therapy in human disease. In this review, we discuss the structure and function of PDEs and examine current and evolving therapeutic uses of PDE inhibitors, highlighting their mechanisms and innovative applications that could further leverage this crucial family of enzymes in clinical settings.

Ligand-binding characteristics of CYP51 Mycobacterium tuberculosis in relation to marine steroid compounds

CYP51 steroid-14α-demethylases are members of a large superfamily of cytochrome P450 enzymes found in all kingdoms of living organisms, and catalyze the 14α-demethylation reaction of a number of natural steroids, including lanosterol, obtusifoliol, and 24,25-dihydrolanosterol. CYP51 are important components of the eukaryotic steroid biosynthetic chain, and thus represent one of the main targets for antifungal therapy. A 14α-demethylase CYP51 homologous gene has also been found in the genome of Mycobacterium tuberculosis. At the same time, M. tuberculosis lacks the de novo pathway for steroid biosynthesis. Conservation of CYP51 among the Mycobacterium genus and colocalization in the genome with 3Fe-4S ferredoxin Rv0763c, which maintains its catalytic activity in vitro, may indirectly indicate the involvement of MTCYP51 in a biochemical process important for mycobacteria. In order to characterize the specificity of the MTCYP51 active site to various compounds of isoprenoid nature, we obtained a highly purified MTCYP51 and, using spectrophotometric titration and surface plasmon resonance methods, studied the interaction of MTCYP51 with steroids from marine organisms obtained in the Pacific Institute of Bioorganic Chemistry of the Far Eastern Branch of the Russian Academy of Sciences. The investigated compounds represent a wide range of evolutionarily ancient isoprenoids. The results showed that MTCYP51 is able to bind structurally diverse steroid derivatives in the active site. The conducted studies suggest the biological role of MTCYP51 for pathogenic mycobacteria, which consists in the binding and possible metabolism of exogenous bioregulatory isoprenoids in vivo.

Development and Optimization of a Bromothymol Blue-Based PLA2 Assay Involving POPC-Based Self-Assemblies.

Phospholipase A2 (PLA2) is a superfamily of phospholipase enzymes that dock at the water/oil interface of phospholipid assemblies, hydrolyzing the ester bond at the sn-2 position. The enzymatic activity of these enzymes differs based on the nature of the substrate, its supramolecular assemblies (micelle, liposomes), and their composition, reflecting the interfacial nature of the PLA2s and requiring assays able to directly quantify this interaction of the enzyme(s) with these supramolecular assemblies. We developed and optimized a simple, universal assay method employing the pH-sensitive indicator dye bromothymol blue (BTB), in which different POPC (3-palmitoyl-2-oleoyl-sn-glycero-1-phosphocholine) self-assemblies (liposomes or mixed micelles with Triton X-100 at different molar ratios) were used to assess the enzymatic activity. We used this assay to perform a comparative analysis of PLA2 kinetics on these supramolecular assemblies and to determine the kinetic parameters of PLA2 isozymes IB and IIA for each supramolecular POPC assembly. This assay is suitable for assessing the inhibition of PLA2s with great accuracy using UV-VIS spectrophotometry, being thus amenable for screening of PLA2 enzymes and their substrates and inhibitors in conditions very similar to physiologic ones.

Characterization of a cytochrome P450 that catalyzes the O-demethylation of lignin-derived benzoates

Cytochromes P450 (P450s) are a superfamily of heme-containing enzymes possessing a broad range of monooxygenase activities. One such activity is O-demethylation, an essential and rate-determining step in emerging strategies to valorize lignin that employ carbon-carbon bond cleavage. We recently identified PbdA, a P450 from Rhodococcus jostii RHA1, and PbdB, its cognate reductase, which catalyze the O-demethylation of para-methoxylated benzoates (p-MBAs) to initiate growth of RHA1 on these compounds. PbdA had the highest affinity (Kd = 3.8 ± 0.6 μM) and apparent specificity (kcat/KM = 20 000 ± 3 000 M-1 s-1) for p-MBA. The enzyme also O-demethylated two related lignin-derived aromatic compounds with remarkable efficiency: veratrate and isovanillate. PbdA also catalyzed the hydroxylation and dehydrogenation of p-EB even though RHA1 did not grow on this compound. Atomic-resolution structures of PbdA in complex with p-MBA, p-EB and veratrate revealed a cluster of three residues that form hydrogen bonds with the substrates’ carboxylate: Ser87, Ser237 and Arg84. Substitution of these residues resulted in lower affinity and O-demethylation activity on p-MBA as well as increased affinity for the acetyl analogue, p-methoxyacetophenone. The S87A and S237A variants of PbdA also catalyzed the O-demethylation of an aldehyde analogue of p-MBA, p-methoxy-benzaldehyde, while the R84M variant did not, despite binding this compound with high affinity. These results suggest that Ser87, Ser237 and Arg84 are not only important determinants of specificity but also help to orientate that substrate correctly in the active site. This study facilitates the design of biocatalysts for lignin valorization.

N-carboxyacyl and N-α-aminoacyl derivatives of aminoaldehydes as shared substrates of plant aldehyde dehydrogenases 10 and 7

Aldehyde dehydrogenases (ALDHs) represent a superfamily of enzymes, which oxidize aldehydes to the corresponding acids. Certain families, namely ALDH9 and ALDH10, are best active with ω-aminoaldehydes arising from the metabolism of polyamines such as 3-aminopropionaldehyde and 4-aminobutyraldehyde. Plant ALDH10s show broad specificity and accept many different aldehydes (aliphatic, aromatic and heterocyclic) as substrates. This work involved the above-mentioned aminoaldehydes acylated with dicarboxylic acids, phenylalanine, and tyrosine. The resulting products were then examined with native ALDH10 from pea and recombinant ALDH7s from pea and maize. This investigation aimed to find a common efficient substrate for the two plant ALDH families. One of the best natural substrates of ALDH7s is aminoadipic semialdehyde carrying a carboxylic group opposite the aldehyde group. The substrate properties of the new compounds were demonstrated by mass spectrometry of the reaction mixtures, spectrophotometric assays and molecular docking. The N-carboxyacyl derivatives were good substrates of pea ALDH10 but were only weakly oxidized by the two plant ALDH7s. The N-phenylalanyl and N-tyrosyl derivatives of 3-aminopropionaldehyde were good substrates of pea and maize ALDH7. Particularly the former compound was converted very efficiently (based on the kcat/Km ratio), but it was only weakly oxidized by pea ALDH10. Although no compound exhibited the same level of substrate properties for both ALDH families, we show that these enzymes may possess more common substrates than expected.

Structural basis for the binding of famotidine, cimetidine, guanidine, and pimagedine with serine protease

Serine proteases are among the important groups of enzymes having significant roles in cell biology. Trypsin is a representative member of the serine superfamily of enzymes, produced by acinar cells of pancreas. It is a validated drug target for various ailments including pancreatitis and colorectal cancer. Premature activation of trypsin is involved in the lysis of pancreatic tissues, which causes pancreatitis. It is also reported to be involved in colorectal carcinoma by activating other proteases, such as matrix metalloproteinase (MMPs). The development of novel trypsin inhibitors with good pharmacokinetic properties could play important roles in pharmaceutical sciences. This study reports the crystal structures of bovine pancreatic trypsin with four molecules; cimetidine, famotidine, pimagedine, and guanidine. These compounds possess binding affinity towards the active site (S1) of trypsin. The structures of all four complexes provided insight of the binding of four different ligands, as well as the dynamics of the active site towards the bind with different size ligands. This study might be helpful in designing of new potent inhibitors of trypsin and trypsin like serine proteases.

Unveiling the importance of the C-terminus in the sugar acid dehydratase of the IlvD/EDD superfamily

Microbial non-phosphorylative oxidative pathways present promising potential in the biosynthesis of platform chemicals from the hemicellulosic fraction of lignocellulose. An L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii catalyzes the rate-limiting step in the non-phosphorylative oxidative pathways, that is, converts sugar acid to 2-dehydro-3-deoxy sugar acid. We have shown earlier that the enzyme forms a dimer of dimers, in which the C-terminal histidine residue from one monomer participates in the formation of the active site of an adjacent monomer. The histidine appears to be conserved across the sequences of sugar acid dehydratases. To study the role of the C-terminus, five variants (H579A, H579F, H579L, H579Q, and H579W) were produced. All variants showed decreased activity for the tested sugar acid substrates, except the variant H579L on D-fuconate, which showed about 20% increase in activity. The reaction kinetic data showed that the substrate preference was slightly modified in H579L compared to the wild-type enzyme, demonstrating that the alternation of the substrate preference of sugar acid dehydratases is possible. In addition, a crystal structure of H579L was determined at 2.4 Å with a product analog 2-oxobutyrate. This is the first enzyme-ligand complex structure from an IlvD/EDD superfamily enzyme. The binding of 2-oxobutyrate suggests how the substrate would bind into the active site in the orientation, which could lead to the dehydration reaction.Key points• Mutation of the last histidine at the C-terminus changed the catalytic activity of L-arabinonate dehydratase from R. leguminosarum bv. trifolii against various C5/C6 sugar acids.• The variant H579L of L-arabinonate dehydratase showed an alteration of substrate preferences compared with the wild type.• The first enzyme-ligand complex crystal structure of an IlvD/EDD superfamily enzyme was solved.Graphical

Identification of long-chain acyl-CoA synthetase gene family reveals that SlLACS1 is essential for cuticular wax biosynthesis in tomato

Long-chain acyl-CoA synthetases (LACSs), belonging to the acyl-activating enzyme superfamily, play crucial roles in lipid biosynthesis and fatty acid catabolism. Here, we identified 11 LACS genes in the tomato reference genome, and these genes were clustered into six subfamilies. Gene structure and conserved motif analyses indicated that LACSs from the same subfamily shared conserved gene and protein structures. Expression analysis revealed that SlLACS1 was highly expressed in the outer epidermis of tomato fruits and leaves. Subcellular localization assay results showed that SlLACS1 was located in the endoplasmic reticulum. Compared with wild-type plants, the wax content on leaves and fruits decreased by 22.5–34.2 % in SlLACS1 knockout lines, confirming that SlLACS1 was involved in wax biosynthesis in both leaves and fruits. Water loss, chlorophyll extraction, water-deficit, and toluidine blue assays suggested that cuticle permeability was elevated in SlLACS1 knockout lines, resulting in reduction in both drought stress resistance and fruit shelf-life. Overall, our analysis of the LACSs in tomato, coupled with investigations of SlLACS1 function, yielded a deeper understanding of the evolutionary patterns of LACS members and revealed the involvement of SlLACS1 in wax accumulation contribute to drought resistance and extended fruit shelf-life in tomato.

Heterocyclic Nitrogen Compounds as Potential PDE4B Inhibitors in Activated Macrophages

Cyclic-nucleotide phosphodiesterases (PDEs) represent a superfamily of enzymes playing a pivotal role in cell signaling by controlling cAMP and cGMP levels in response to receptor activation. PDE activity and expression are linked to many diseases including inflammatory diseases. In light of their specific biochemical properties, PDE inhibition has attracted the interest of several researrs In this context, PDE4 inhibition induces anti-inflammatory effects. Piclamilast and rolipram, well-known PDE4 inhibitors, are endowed with common side effects. The selective phosphodiesterase 4B (PDE4B) inhibitors could be a promising approach to overcome these side effects. In the present study, six potential PDE4B inhibitors have been investigated. Through this study, we identified three PDE4B inhibitors in human macrophages activated by lipopolysaccharide. Interestingly, two of them reduced reactive oxygen species production in pro-inflammatory macrophages.

Arsinothricin Biosynthesis Involving a Radical SAM Enzyme for Noncanonical SAM Cleavage and C-As Bond Formation.

Arsinothricin is a potent antibiotic secreted by soil bacteria. The biosynthesis of arsinothricin was proposed to involve a C-As bond formation between trivalent As and the 3-amino-3-carboxypropyl (ACP) group of S-adenosyl-l-methionine (SAM), which is catalyzed by the protein ArsL. However, ArsL has not been characterized in detail. Interestingly, ArsL contains a CxxxCxxC motif and thus belongs to the radical SAM enzyme superfamily, the members of which cleave SAM and generate a 5'-deoxyadenosyl radical. Here, we found that ArsL cleaves the Cγ,Met-S bond of SAM and generates an ACP radical that resembles Dph2, a noncanonical radical SAM enzyme involved in diphthamid biosynthesis. As Dph2 does not contain the CxxxCxxC motif, ArsL is a unique radical SAM enzyme that contains this motif but generates a noncanonical ACP radical. Together with the methyltransferase ArsM, we successfully reconstituted arsinothricin biosynthesis in vitro. ArsL has a conserved RCCLKC motif in the C-terminal sequence and belongs to the RCCLKC-tail radical SAM protein subfamily. By truncation and mutagenesis, we showed that this motif plays an important role in binding to the substrate arsenite and is highly important for its activity. Our results suggested that ArsL has a canonical radical SAM enzyme motif but catalyzes a noncanonical radical SAM reaction, implying that more noncanonical radical SAM chemistry may exist within the radical SAM enzyme superfamily.

Computational stabilization of a non-heme iron enzyme enables efficient evolution of new function.

Directed evolution has emerged as a powerful tool for engineering new biocatalysts. However, introducing new catalytic residues can be destabilizing, and it is generally beneficial to start with a stable enzyme parent. Here we show that the deep learning-based tool ProteinMPNN can be used to redesign Fe(II)/αKG superfamily enzymes for greater stability, solubility, and expression while retaining both native activity and industrially-relevant non-native functions. For the Fe(II)/αKG enzyme tP4H, we performed site-saturation mutagenesis with both the wild-type and stabilized design variant and screened for activity increases in a non-native C-H hydroxylation reaction. We observed substantially larger increases in non-native activity for variants obtained from the stabilized scaffold compared to those from the wild-type enzyme. ProteinMPNN is user-friendly and widely-accessible, and straightforward structural criteria were sufficient to obtain stabilized, catalytically-functional variants of the Fe(II)/αKG enzymes tP4H and GriE. Our work suggests that stabilization by computational sequence redesign could be routinely implemented as a first step in directed evolution campaigns for novel biocatalysts.