Search

Protein kinases play crucial roles in regulating cellular processes, making real-time visualization of their activity essential for understanding signaling dynamics. While genetically encoded fluorescent biosensors have emerged as powerful tools for studying kinase activity, their development for many kinases remains challenging due to the lack of suitable substrate peptides. Here, we present a novel approach for identifying peptide substrates and demonstrate its effectiveness by developing a biosensor for Protein Kinase N (PKN) activity. Our method identified a new PKN substrate peptide that we optimized for use in a fluorescent biosensor design. The resulting biosensor shows specificity for PKN family kinases and can detect both overexpressed and endogenous PKN activity in live cells. Importantly, our biosensor revealed sustained basal PKN2 activity at the plasma membrane, identifying it as a PKN2 activity hotspot. This work not only provides a valuable tool for studying PKN signaling but also demonstrates a promising strategy for developing biosensors for other understudied kinases, potentially expanding our ability to monitor kinase activity across the human kinome.

DNA methylation is an important epigenetic mechanism essential for transposon silencing and genome integrity. Across evolution, the substrates of DNA methylation have diversified between kingdoms. In plants, chromomethylase3 (CMT3) and CMT2 mediate CHG and CHH methylation, respectively. However, how these two methyltransferases diverge on substrate specificities during evolution remains unknown. Here, we reveal that CMT2 originates from a duplication of an evolutionarily ancient CMT3 in flowering plants. Lacking a key arginine residue recognizing CHG in CMT2 impairs its CHG methylation activity in most flowering plants. An engineered V1200R mutation empowers CMT2 to restore CHG and CHH methylations in Arabidopsis cmt2cmt3 mutant, testifying a loss-of-function effect for CMT2 during evolution. CMT2 has evolved a long and unstructured amino terminus critical for protein stability, especially under heat stress, and is plastic to tolerate various natural mutations. Together, this study reveals the mechanism of chromomethylase divergence for context-specific DNA methylation in plants and sheds important lights on DNA methylation evolution and function.

DNA methylation is one of the major epigenetic mechanisms crucial for gene regulation and genome stability. De novo DNA methyltransferase DNMT3C is required for silencing evolutionarily young transposons during mice spermatogenesis. Mutation of DNMT3C led to a sterility phenotype that cannot be rescued by its homologs DNMT3A and DNMT3B. However, the structural basis of DNMT3C-mediated DNA methylation remains unknown. Here, we report the structure and mechanism of DNMT3C-mediated DNA methylation. The DNMT3C methyltransferase domain recognizes CpG-containing DNA in a manner similar to that of DNMT3A and DNMT3B, in line with their high sequence similarity. However, two evolutionary covariation sites, C543 and E590, diversify the substrate interaction among DNMT3C, DNMT3A, and DNMT3B, resulting in distinct DNA methylation activity and specificity between DNMT3C, DNMT3A, and DNMT3B in vitro. In addition, our combined structural and biochemical analysis reveals that the disease-causing rahu mutation of DNMT3C compromises its oligomerization and DNA-binding activities, explaining the loss of DNA methylation activity caused by this mutation. This study provides a mechanistic insight into DNMT3C-mediated DNA methylation that complements DNMT3A- and DNMT3B-mediated DNA methylation in mice, unraveling a regulatory mechanism by which evolutionary conservation and diversification fine-tune the activity of de novo DNA methyltransferases.

Glycosylation is a predominant strategy plants use to fine-tune the properties of small molecule metabolites to affect their bioactivity, transport, and storage. It is also important in biotechnology and medicine as many glycosides are utilized in human health. Small molecule glycosylation is largely carried out by family 1 glycosyltransferases. Here, we report a structural and biochemical investigation of UGT95A1, a family 1 GT enzyme from Pilosella officinarum that exhibits a strong, unusual regiospecificity for the 3'-O position of flavonoid acceptor substrate luteolin. We obtained an apo crystal structure to help drive the analyses of a series of binding site mutants, revealing that while most residues are tolerant to mutations, key residues M145 and D464 are important for overall glycosylation activity. Interestingly, E347 is crucial for maintaining the strong preference for 3'-O glycosylation, while R462 can be mutated to increase regioselectivity. The structural determinants of regioselectivity were further confirmed in homologous enzymes. Our study also suggests that the enzyme contains large, highly dynamic, disordered regions. We showed that while most disordered regions of the protein have little to no implication in catalysis, the disordered regions conserved among investigated homologs are important to both the overall efficiency and regiospecificity of the enzyme. This report represents a comprehensive in-depth analysis of a family 1 GT enzyme with a unique substrate regiospecificity and may provide a basis for enzyme functional prediction and engineering.

Aminotransferases (ATs) are an ancient enzyme family that play central roles in core nitrogen metabolism, essential to all organisms. However, many of the AT enzyme functions remain poorly defined, limiting our fundamental understanding of the nitrogen metabolic networks that exist in different organisms. Here, we traced the deep evolutionary history of the AT family by analyzing AT enzymes from 90 species spanning the tree of life (ToL). We found that each organism has maintained a relatively small and constant number of ATs. Mapping the distribution of ATs across the ToL uncovered that many essential AT reactions are carried out by taxon-specific AT enzymes due to wide-spread nonorthologous gene displacements. This complex evolutionary history explains the difficulty of homology-based AT functional prediction. Biochemical characterization of diverse aromatic ATs further revealed their broad substrate specificity, unlike other core metabolic enzymes that evolved to catalyze specific reactions today. Interestingly, however, we found that these AT enzymes that diverged over billion years share common signatures of multisubstrate specificity by employing different nonconserved active site residues. These findings illustrate that AT family enzymes had leveraged their inherent substrate promiscuity to maintain a small yet distinct set of multifunctional AT enzymes in different taxa. This evolutionary history of versatile ATs likely contributed to the establishment of robust and diverse nitrogen metabolic networks that exist throughout the ToL. The study provides a critical foundation to systematically determine diverse AT functions and underlying nitrogen metabolic networks across the ToL.

Crystal structures of human long-chain acyl-CoA dehydrogenase (LCAD) and the catalytically inactive Glu291Gln mutant, have been determined. These structures suggest that LCAD harbors functions beyond its historically defined role in mitochondrial β-oxidation of long and medium-chain fatty acids. LCAD is a homotetramer containing one FAD per 43 kDa subunit with Glu291 as the catalytic base. The substrate binding cavity of LCAD reveals key differences which makes it specific for longer and branched chain substrates. The presence of Pro132 near the start of the E helix leads to helix unwinding that, together with adjacent smaller residues, permits binding of bulky substrates such as 3α, 7α, l2α-trihydroxy-5β-cholestan-26-oyl-CoA. This structural element is also utilized by ACAD11, a eucaryotic ACAD of unknown function, as well as bacterial ACADs known to metabolize sterol substrates. Sequence comparison suggests that ACAD10, another ACAD of unknown function, may also share this substrate specificity. These results suggest that LCAD, ACAD10, ACAD11 constitute a distinct class of eucaryotic acyl CoA dehydrogenases.

Insertion sequence (IS) elements are the simplest autonomous transposable elements found in prokaryotic genomes1. We recently discovered that IS110 family elements encode a recombinase and a non-coding bridge RNA (bRNA) that confers modular specificity for target DNA and donor DNA through two programmable loops2. Here we report the cryo-electron microscopy structures of the IS110 recombinase in complex with its bRNA, target DNA and donor DNA in three different stages of the recombination reaction cycle. The IS110 synaptic complex comprises two recombinase dimers, one of which houses the target-binding loop of the bRNA and binds to target DNA, whereas the other coordinates the bRNA donor-binding loop and donor DNA. We uncovered the formation of a composite RuvC-Tnp active site that spans the two dimers, positioning the catalytic serine residues adjacent to the recombination sites in both target and donor DNA. A comparison of the three structures revealed that (1) the top strands of target and donor DNA are cleaved at the composite active sites to form covalent 5-phosphoserine intermediates, (2) the cleaved DNA strands are exchanged and religated to create a Holliday junction intermediate, and (3) this intermediate is subsequently resolved by cleavage of the bottom strands. Overall, this study reveals the mechanism by which a bispecific RNA confers target and donor DNA specificity to IS110 recombinases for programmable DNA recombination.

SLC22A10 is an orphan transporter with unknown substrates and function. The goal of this study is to elucidate its substrate specificity and functional characteristics. In contrast to orthologs from great apes, human SLC22A10, tagged with green fluorescent protein, is not expressed on the plasma membrane. Cells expressing great ape SLC22A10 orthologs exhibit significant accumulation of estradiol-17β-glucuronide, unlike those expressing human SLC22A10. Sequence alignments reveal a proline at position 220 in humans, which is a leucine in great apes. Replacing proline with leucine in SLC22A10-P220L restores plasma membrane localization and uptake function. Neanderthal and Denisovan genomes show proline at position 220, akin to modern humans, indicating functional loss during hominin evolution. Human SLC22A10 is a unitary pseudogene due to a fixed missense mutation, P220, while in great apes, its orthologs transport sex steroid conjugates. Characterizing SLC22A10 across species sheds light on its biological role, influencing organism development and steroid homeostasis.

Dipeptide production mediated by dipeptidyl-peptidase (DPP)4, DPP5, DPP7, and DPP11 plays a crucial role in growth of Porphyromonas gingivalis, a periodontopathic asaccharolytic bacterium. Given the particular P1-position specificity of DPPs, it has been speculated that DPP5 or DPP7 might be responsible for degrading refractory P1 amino acids, i.e., neutral (Thr, His, Gly, Ser, Gln) and hydrophilic (Asn) residues. The present results identified DPP7 as an entity that processes these residues, thus ensuring complete production of nutritional dipeptides in the bacterium. Activity enhancement by the P1′ residue was observed in DPP7, as well as DPP4 and DPP5. Toward the refractory P1 residues, DPP7 uniquely hydrolyzed HX|LD-MCA (X = His, Gln, or Asn) and their hydrolysis was most significantly suppressed in dpp7 gene-disrupted cells. Additionally, hydrophobic P2 residue significantly enhanced DPP7 activity toward these substrates. The findings propose a comprehensive 20 P1 × 20 P2 amino acid matrix showing the coordination of four DPPs to achieve complete dipeptide production along with subsidiary peptidases. The present finding of a broad substrate specificity that DPP7 accounts for releasing 48 % (192/400) of N-terminal dipeptides could implicate its potential role in linking periodontopathic disease to related systemic disorders. [ABSTRACT FROM AUTHOR]

The purified E.coil-derived patatin is an enzyme with a molecular mass of 40 kDa, it displayed optimal esterase activity (against p-Nitrophenyl acetate, p-NPC2) at 37 °C and a pH of 9.0 for 25 min. Its enzymatic activity was stable at pH 4.0–9.0 and could retain about 65.8% of its activity at 90 °C for 1 h. The residual activity remained over 80% in 4 weeks' duration when patatin was freeze-dried or stored in a buffer solution of -80 to 25 °C, the residual activity remained over 80% for 4 weeks. Patatin's enzymatic hydrolysis ability against p-NPC2 was enhanced in the presence of K+, Mg2+, Al3+. Most organic solvents had no significant effects on its activity except ethyl acetate. Patatin showed broad specificity to substrates of different carbon chain lengths (C2-C16), of which the highest was against short carbon chains (C2-C4), with the lowest Km of 1.01 mM and the highest Vmax of 49.75 mmol/L·min to p-NPC2. The patatin also possessed remarkable lipase activity in hydrolyzing oils and high selectivity to animal fats. These results suggested that the recombinant patatin has promising marketable potential in the hydrolysis of important ester or lipid in food, pharmaceutical, biochemical, and biological interests due to its broad substrate specificity and high stability. [ABSTRACT FROM AUTHOR]

Quercetin, naringenin, and hesperetin are the hydrolysis products of rutinoside flavonoids, known for their multiple biological activities and potential applications. To explore the characteristics of different rutinosidases in preparing quercetin, naringenin, and hesperetin, the rutinosidase AnRut from Aspergillus niger CBS 513.88, and αRβD I and αRβD II from Acremonium sp. DSM 24697 were selected for comparative studies on substrate specificity and enzymatic properties. The results showed that, all three rutinosidases could only hydrolyze flavonoid compounds with α-1,6-linked rutinosides, but had no effect on flavonoid compounds with α-1,2-linked neohesperidosides. AnRut primarily hydrolyzed 3-O-linked rutin, αRβD I mainly hydrolyzed 7-O-linked narirutin and hesperidin, while αRβD II showed no significant difference in hydrolytic activity towards both types of substrates. Molecular docking results indicated that there were distinct binding modes within the three rutinosidases with rutin, narirutin, and hesperidin, and the substrate specificities of the three rutinosidases were influenced with variations in their interactions with the glycoside structures rutinoside flavonoids. The optimal temperature for AnRut was 50 °C, and the optimal pH was 4.0. Additionally, 10 mmol/L β-ME and DTT significantly enhanced AnRut's enzymatic activity, increasing the relative activity to 223% and 242% of the wild type, respectively. The optimum temperature and pH of αRβD I was 70 °C and 4.0, demonstrating efficient hydrolysis of narirutin and hesperidin under acidic conditions. αRβD II, meanwhile, had an optimal temperature of 40 t and an optimal pH of 6.0, indicating its suitability for hydrolyzing rutinoside flavonoids under neutral conditions. This study would provide experimental and theoretical references for the preparation of quercetin, naringenin, and hesperetin using rutinosidases and lay the groundwork for future research on the structure-activity relationship of rutinosidases. [ABSTRACT FROM AUTHOR]

Lipases are effective clean-label improvers for the baking quality of cake. Insights into lipase activities in different cake formulations in combination with the effect on batter/dough and baking quality are needed to further reveal the underlying mechanisms. Therefore, a method using normal phase high-performance liquid chromatography coupled to an evaporative light scattering detector was adapted and validated for the five most abundant lipid classes in three common cake recipes, namely triacylglycerols, diacylglycerols, monoacylglycerols, glycerophosphocholine and lysoglycerophosphocholine. The method revealed total changes of 0.2 mg/g to 186.5 mg/g in lipid class content per dry weight after lipase treatments. Comparative investigations on batter/dough and products of basic cake, pound cake and brioche without or with addition of seven lipases showed that the substrate specificity of lipases is the decisive factor for their effectiveness regarding improved dough and product quality. A high lipase activity only supported already matching substrate specificities. [ABSTRACT FROM AUTHOR]

To reduce carbon emissions and address environmental concerns, the aviation industry is exploring the use of sustainable aviation fuel (SAF) as an alternative to traditional fossil fuels. In this context, bio‐alkane is considered a potentially high‐value solution. The present study focuses on the enzymes acyl‐acyl carrier protein [ACP] reductase (AAR) and aldehyde‐deformylating oxygenase (ADO), which are crucial enzymes for alka(e)ne biosynthesis. By using protein engineering techniques, including semi‐rational design and site‐directed mutagenesis, we aimed to enhance the substrate specificity of AAR and improve alkane production efficiency. The co‐expression of a modified AAR (Y26G/Q40M mutant) with wild‐type ADO in Escherichia coli significantly increased alka(e)ne production from 28.92 mg/L to 167.30 mg/L, thus notably demonstrating a 36‐fold increase in alkane yield. This research highlights the potential of protein engineering in optimizing SAF production, thereby contributing to the development of more sustainable and efficient SAF production methods and promoting greener air travel. [ABSTRACT FROM AUTHOR]

Glucose‐6‐phosphatase‐β (G6PC3) is a ubiquitous phosphatase present in the endoplasmic reticulum, which, unlike G6PC1, is not responsible for maintaining blood glucose level under starvation. Recently, G6PC3 has been shown to play an important role in neutrophil granulocytes, eliminating the toxic metabolite 1,5‐anhydroglucitol‐6‐phosphate. The present study aimed to look for alternative substrates for the enzyme and outline the expression changes in the parts of this multicomponent system during neutrophil granulocyte differentiation. We determined the kinetic characteristics of recombinant human G6PC3 towards different sugar phosphates, and the transport of these compounds was also measured in rat liver microsomes. We found that all investigated sugar phosphates are substrates for G6PC3, although their microsomal transport is much slower than that of glucose‐6‐phosphate. Using the HL‐60 promyelocytic leukemia cell line as an in vitro model system for myeloid differentiation, we found no significant differences in enzyme expression and phosphatase activity latency between undifferentiated and differentiated cells. Our results provide novel insights into the possible role of G6PC3 in the dephosphorylation of alternative sugar phosphates or their metabolites synthesized in the endoplasmic reticulum and confirm the potential feature of the enzyme in the promyelocytic stage as well. These findings contribute to our knowledge of intracellular carbohydrate metabolism of neutrophil granulocytes, which facilitates further research directions to better understand the underlying mechanisms of neutropenias.

Quercetin, naringenin, and hesperetin are the hydrolysis products of rutinoside flavonoids, known for their multiple biological activities and potential applications. To explore the characteristics of different rutinosidases in preparing quercetin, naringenin, and hesperetin, the rutinosidase AnRut from Aspergillus niger CBS 513.88, and αRβD I and αRβD II from Acremonium sp. DSM 24697 were selected for comparative studies on substrate specificity and enzymatic properties. The results showed that, all three rutinosidases could only hydrolyze flavonoid compounds with α-1,6-linked rutinosides, but had no effect on flavonoid compounds with α-1,2-linked neohesperidosides. AnRut primarily hydrolyzed 3-O-linked rutin, αRβD I mainly hydrolyzed 7-O-linked narirutin and hesperidin, while αRβD II showed no significant difference in hydrolytic activity towards both types of substrates. Molecular docking results indicated that there were distinct binding modes within the three rutinosidases with rutin, narirutin, and hesperidin, and the substrate specificities of the three rutinosidases were influenced with variations in their interactions with the glycoside structures rutinoside flavonoids. The optimal temperature for AnRut was 50 ℃, and the optimal pH was 4.0. Additionally, 10 mmol/L β-ME and DTT significantly enhanced AnRut's enzymatic activity, increasing the relative activity to 223% and 242% of the wild type, respectively. The optimum temperature and pH of αRβD I was 70 ℃ and 4.0, demonstrating efficient hydrolysis of narirutin and hesperidin under acidic conditions. αRβD II, meanwhile, had an optimal temperature of 40 ℃ and an optimal pH of 6.0, indicating its suitability for hydrolyzing rutinoside flavonoids under neutral conditions. This study would provide experimental and theoretical references for the preparation of quercetin, naringenin, and hesperetin using rutinosidases and lay the groundwork for future research on the structure-activity relationship of rutinosidases.

Many peptide hormones form an α-helix on binding their receptors1-4, and sensitive methods for their detection could contribute to better clinical management of disease5. De novo protein design can now generate binders with high affinity and specificity to structured proteins6,7. However, the design of interactions between proteins and short peptides with helical propensity is an unmet challenge. Here we describe parametric generation and deep learning-based methods for designing proteins to address this challenge. We show that by extending RFdiffusion8 to enable binder design to flexible targets, and to refining input structure models by successive noising and denoising (partial diffusion), picomolar-affinity binders can be generated to helical peptide targets by either refining designs generated with other methods, or completely de novo starting from random noise distributions without any subsequent experimental optimization. The RFdiffusion designs enable the enrichment and subsequent detection of parathyroid hormone and glucagon by mass spectrometry, and the construction of bioluminescence-based protein biosensors. The ability to design binders to conformationally variable targets, and to optimize by partial diffusion both natural and designed proteins, should be broadly useful.

Repurposing the intrinsic properties of natural enzymes can offer a viable solution to current synthetic challenges through the development of novel biocatalytic processes. Although amino acid racemases are ubiquitous in living organisms, an amine racemase (AR) has not yet been discovered despite its synthetic potential for producing chiral amines. Here, we report the creation of an AR based on the serendipitous discovery that amine transaminases (ATAs) can perform stereoinversion of 2-aminobutane. Kinetic modeling revealed that the unexpected off-pathway activity results from stereochemically promiscuous futile cycles due to incomplete stereoselectivity for 2-aminobutane. This finding motivated us to engineer an S-selective ATA through in silico alanine scanning and empirical combinatorial mutations, creating an AR with broad substrate specificity. The resulting AR, carrying double point mutations, enables the racemization of both enantiomers of diverse chiral amines in the presence of a cognate ketone. This strategy may be generally applicable to a wide range of transaminases, paving the way for the development of new-to-nature racemases.

Protein homeostasis is a tightly conserved process that is regulated through the ubiquitin proteasome system (UPS) in a ubiquitin-independent or ubiquitin-dependent manner. Over the past two decades, the proteasome has become an excellent therapeutic target through inhibition of the catalytic core particle, inhibition of subunits responsible for recognizing and binding ubiquitinated proteins, and more recently, through targeted protein degradation using proteolysis targeting chimeras (PROTACs). The majority of the developed inhibitors of the proteasome's core particle rely on gaining selectivity through binding interactions within the unprimed substrate channel. Although this has allowed for selective inhibitors and chemical probes to be generated for the different proteasome isoforms, much remains unknown about the interactions that could be harnessed within the primed substrate channel to increase potency or selectivity. Herein, we discuss small molecules that interact with the primed substrate pocket and how their differences may give rise to altered activity. Taking advantage of additional interactions with the primed substrate pocket of the proteasome could allow for the generation of improved chemical tools for perturbing or monitoring proteasome activity.

DAF‐2, the Caenorhabditis elegans insulin‐like receptor homolog, regulates larval development, metabolism, stress response, and lifespan. The availability of numerous daf‐2 mutant alleles has made it possible to elucidate the genetic mechanisms underlying these physiological processes. The DAF‐2 pathway is significantly conserved with the human insulin/IGF‐1 signaling pathway; it includes proteins homologous to human IRS, GRB‐2, and PI3K, making it an important model to investigate human pathological conditions. We expressed and purified the kinase domain of wild‐type DAF‐2 to examine the catalytic activity and substrate specificity of the enzyme. Like the human insulin receptor kinase, DAF‐2 kinase phosphorylates tyrosines within specific YxN or YxxM motifs, which are important for recruiting downstream effectors. DAF‐2 kinase phosphorylated peptides derived from the YxxM and YxN motifs located in the C‐terminal extension of the receptor tyrosine kinase, consistent with the idea that the DAF‐2 receptor may possess independent signaling capacity. Unlike the human insulin or IGF‐1 receptor kinases, DAF‐2 kinase was poorly inhibited by the small‐molecule inhibitor linsitinib. We also expressed and purified mutant kinases corresponding to daf‐2 alleles that result in partial loss‐of‐function phenotypes in C. elegans. These mutations caused a complete loss of kinase function in vitro. Our biochemical investigations provide new insights into DAF‐2 kinase function, and the approach should be useful for studying other mutations to shed light on DAF‐2 signaling in C. elegans physiology. [ABSTRACT FROM AUTHOR]

Copyright of Food & Fermentation Industries is the property of Food & Fermentation Industries and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Acetohydroxyacid synthase (AHAS, EC 2.2.1.6) catalyzes the first step in the synthesis of the branched-chain amino acids valine, leucine, and isoleucine, pathways being present in plants and microorganisms, but not in animals. Thus, AHAS is an important target for numerous herbicides and, more recently, for the development of antimicrobial agents. The need to develop new and optimized herbicides and pharmaceuticals requires a detailed understanding of the biochemistry of AHAS. AHAS transfers an activated two-carbon moiety derived from pyruvate to either pyruvate or 2-oxobutyrate as acceptor substrates, forming 2-acetolactate or 2-acetohydroxy-2-butyrate, respectively. Various methods have been described in the literature to biochemically characterize AHAS with respect to substrate preferences, substrate specificity, or kinetic parameters. However, the simultaneous detection and quantification of substrates and unstable products of the AHAS-catalyzed reaction have always been a challenge. Using AHAS isoform II from Escherichia coli, we have developed a sensitive assay for AHAS-catalyzed reactions that uses derivatization with ethyl chloroformate to stabilize and volatilize all reactants in the aqueous solution and detect them by gas chromatography coupled to flame ionization detection or mass spectrometry. This assay allows us to characterize the product formation in reactions in single and dual substrate reactions and the substrate specificity of AHAS, and to reinterpret previous biochemical observations. This assay is not limited to the AHAS-catalyzed reactions, but should be applicable to studies of many metabolic pathways. [ABSTRACT FROM AUTHOR]

PON1 is a Ca2+-dependent enzyme that indicates a hydrolytic activity towards a broad spectrum of substrates. The mechanism of hydrolysis catalyzed by this enzyme is poorly understood. It was shown that the active site of PON1 is highly dynamic. The catalytic center of this enzyme consists of side chains of amino acids binding two calcium ions, from which the first one performs a structural function and the other one is responsible for the catalytic properties of PON1. This review summarizes available information on the structure of PONs, the role of amino acids located in the active site in specificity, and multiple substrate affinity of enzymes for understanding and explaining the basis of the physiological function of PONs. Moreover, in this paper, we described the changes in the structure of PONs induced by environmental and genetic factors and their association with diseases. The detoxification efficiency depends on the polymorphism of the PON1 gene, especially Q192R. However, data on the association between single-nucleotide polymorphisms (SNPs) in the PON1 gene and cardiovascular or neurodegenerative diseases are insufficient. The reviewed papers may confirm that PON1 is a very promising tool for diagnostics, but further studies are required. [ABSTRACT FROM AUTHOR]

Gut microbes produce α- l -fucosidases critical for utilizing human milk oligosaccharides, mucosal and dietary glycans. Although gut Parabacteroides have garnered attention for their impact on host health and disease, their CAZymes remain poorly studied. CAZome analysis of eleven gut Parabacteroides type strains revealed their capacity to degrade mucin O -glycans. Their abundance of GH29 fucosidases caught our attention, and we predicted the functional profiles of 46 GH29 fucosidases using in silico approaches. Our findings showed diverse linkages specificities and species-specific distributions, with over half of GH29 enzymes functioning as α1,3/4 fucosidases, essential for acting on Lewis antigen epitopes of mucin O -glycans. We further enzymatically validated 4 novel GH29 sequences from poorly characterized groups. PgoldGH29A (cluster37GH29BERT, GH29:75.1CUPP) does not act on tested natural substrates. PgoldGH29B (cluster1GH29BERT, GH29:84.1CUPP) functions as a strict α1,3/4 fucosidase. PgoldGH29C (cluster14GH29BERT, GH29:29.1CUPP) displays unprecedented substrate specificity for α1,2/3/4 disaccharides. PgoldGH29D (cluster4GH29BERT, GH29:6.2CUPP) acts on α1,2/3/4/6 linkages similar to enzymes from GH29:6.1CUPP but prefers disaccharides over trisaccharides. These results suggest that PgoldGH29B and PgoldGH29D can contribute to mucin O -glycan degradation via their α1,3/4 and α1,2 fucosidase activity, respectively, while the natural substrates of PgoldGH29A and PgoldGH29C may be irrelevant to host-glycans. These insights enhance our understanding of the ecological niches inhabited by gut Parabacteroides and may guide similar exploration in other intriguing gut microbial species. [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases (LPMOs) are one of the emerging classes of copper metalloenzymes that have received considerable attention due to their ability to boost the enzymatic conversion of intractable polysaccharides such as plant cell walls and chitin polymers. LPMOs catalyze the oxidative cleavage of β‐1,4‐glycosidic bonds using molecular O2 or H2O2 in the presence of an external electron donor. LPMOs have been classified as an auxiliary active (AA) class of enzymes and, further based on substrate specificity, divided into eight families. Until now, multiple LPMOs from AA9 and AA10 families, mostly from microbial sources, have been investigated; the exact mechanism and structure–function are elusive to date, and recently discovered AA families of LPMOs are just scratched. This review highlights the origin and discovery of the enzyme, nomenclature, three‐dimensional protein structure, substrate specificity, copper‐dependent reaction mechanism, and different techniques used to determine the product formation through analytical and biochemical methods. Moreover, the diverse functions of proteins in various biological activities such as plant–pathogen/pest interactions, cell wall remodeling, antibiotic sensitivity of biofilms, and production of nanocellulose along with certain obstacles in deconstructing the complex polysaccharides have also been summarized, while highlighting the innovative and creative ways to overcome the limitations of LPMOs in hydrolyzing the biomass. [ABSTRACT FROM AUTHOR]

Copyright of China Brewing is the property of China Brewing Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Activation of fatty acids as acyl-adenylates by fatty acid-AMP ligase (FAAL) is a well-established process contributing to the formation of various functional natural products. Enzymatic characterization of FAALs is pivotal for unraveling both the catalytic mechanism and its role in specific biosynthetic pathways. In this study, we recombinantly expressed and characterized a novel FAAL derived from marine Pseudoalteromonas citrea (PcFAAL). PcFAAL was a cold-adapted neutral enzyme, demonstrating optimal activity at 30 °C and pH 7.5. Notably, its specific activity relied on the presence of Mg2+; however, higher concentrations exceeding 10 mM resulted in inhibition of enzyme activity. Various organic solvents, especially water-immiscible organic solvents, demonstrated an activating effect on the activity of PcFAAL on various fatty acids. The specific activity exhibited a remarkable 50-fold increase under 4% (v/v) n-hexane compared to the aqueous system. PcFAAL displayed a broad spectrum of fatty acid substrate selectivity, with the highest specific activity for octanoic acid (C8:0), and the catalytic efficiency (kcat/Km) for octanoic acid was determined to be 1.8 nM−1·min−1. Furthermore, the enzyme demonstrated biocatalytic promiscuity in producing a class of N-acyl amino acid natural products, as verified by LC-ESI MS. Results indicated that the PcFAAL exhibits promiscuity towards 10 different kinds of amino acids and further demonstrated their potential value in the biosynthesis of corresponding functional N-acyl amino acids. [ABSTRACT FROM AUTHOR]

The protozoan parasite Trichomonas vaginalis (Tv) causes trichomoniasis, the most common non‐viral sexually transmitted infection in the world. Although Tv has been linked to significant health complications, only two closely related 5‐nitroimidazole drugs are approved for its treatment. The emergence of resistance to these drugs and lack of alternative treatment options poses an increasing threat to public health, making development of novel anti‐Trichomonas compounds an urgent need. The proteasome, a critical enzyme complex found in all eukaryotes has three catalytic subunits, β1, β2, and β5 and has been validated as a drug target to treat trichomoniasis. With the goal of developing tools to study the Tv proteasome, we isolated the enzyme complex and identified inhibitors that preferentially inactivate either one or two of the three catalytic subunits. Using a mass spectrometry‐based peptide digestion assay, these inhibitors were used to define the substrate preferences of the β1, β2 and β5 subunits. Subsequently, three model fluorogenic substrates were designed, each specific for one of the catalytic subunits. This novel substrate profiling methodology will allow for individual subunit characterization of other proteasomes of interest. Using the new substrates, we screened a library of 284 peptide epoxyketone inhibitors against Tv and determined the subunits targeted by the most active compounds. The data show that inhibition of the Tv β5 subunit alone is toxic to the parasite. Taken together, the optimized proteasome subunit substrates will be instrumental for understanding the molecular determinants of proteasome specificity and for accelerating drug development against trichomoniasis. [ABSTRACT FROM AUTHOR]

Bacterial sortases are a family of cysteine transpeptidases in Gram‐positive bacteria of which sortase A (SrtA) enzymes are responsible for ligating proteins to the peptidoglycan layer of the cell surface. Engineered versions of sortases are also used in sortase‐mediated ligation (SML) strategies for a variety of protein engineering applications. Although a versatile tool, substrate recognition by Staphylococcus aureus SrtA (saSrtA), the most commonly utilized enzyme in SML, is stringent and relies on an LPXTG pentapeptide motif. Previous structural studies revealed that the requirement of a glycine in the binding motif may be due to potential steric hindrance of amino acids possessing a β‐carbon by W194, a tryptophan located in the β7‐β8 loop of the enzyme. Here, we measured the effect of seven single point mutants of W194 (A, D, F, G, N, S, Y) saSrtA using a FRET‐based activity assay. We found that while the LPXTG motif remains a requirement for initial proteolytic cleavage, the nucleophile specificity of our variants is altered. In particular, W194A and W194S saSrtA recognize a D‐Ala nucleophile and are able to perform ligation reactions. Notably, an LPXT(D‐Ala) peptide was not cleaved by either mutant enzyme. We hypothesize that these variants may potentially be utilized to develop an irreversible sortase‐mediated reaction. Taken together, this experiment reveals new insight into sortase specificity and possible future SML strategies. [ABSTRACT FROM AUTHOR]

ATP-binding cassette (ABC) transporters, particularly those of subfamily B, are involved in cell detoxification, multidrug resistance, drug treatment pharmacodynamics, and also ecological adaptation. In this regard, ABCB transporters may play a decisive role in the co-evolution between plants and herbivores. Cardenolides, toxic steroid glycosides, are secondary plant metabolites that defend plants against herbivores by targeting their sodium–potassium ATPase. Despite their toxicity, several herbivorous insects such as the large milkweed bug (Oncopeltus fasciatus) have evolved adaptations to tolerate cardenolides and sequester them for their own defense. We investigate the role of two ABCB transporters of O. fasciatus for the paracellular transport of cardenolides by docking simulations and ATPase assays. Cardenolide binding of OfABCB1 and OfABCB2 is predicted by docking simulations and calculated binding energies are compared with substrate specificities determined in ATPase assays. Both tested ABCB transporters showed activity upon exposure to cardenolides and Km values that agreed well with the predictions of our docking simulations. We conclude that docking simulations can help identify transporter binding regions and predict substrate specificity, as well as provide deeper insights into the structural basis of ABC transporter function. [ABSTRACT FROM AUTHOR]

β-Galactosidase from Bacillus circulans ATCC 31382 (BgaD) is a biotechnologically important enzyme for the synthesis of β-galactooligosaccharides (GOS). Among its four isoforms, isoform A (BgaD-A) has distinct synthetic properties. Here, we present cryoelectron microscopy (cryo-EM) structures of BgaD-A and compare them with the known X-ray crystal structure of isoform D (BgaD-D), revealing substantial structural divergences between the two isoforms. In contrast to BgaD-D, BgaD-A features a flexible Big-4 domain and another enigmatic domain. The newly identified flexible region in BgaD-A is termed as "barrier domain 8," and serves as a barricade, obstructing the access of longer oligosaccharide substrates into the active site of BgaD-A. The transgalactosylation reactions catalyzed by both isoforms revealed that BgaD-A has a higher selectivity than BgaD-D in the earlier stages of the reaction and is prevailingly directed to shorter galactooligosaccharides. This study improves our understanding of the structural determinants governing β-galactosidase catalysis, with implications for tailored GOS production. [Display omitted] • BgaD-A cryo-EM structures revealed structural differences between BgaD isoforms • BgaD-A contains a "Barrier domain 8" that is lacking in BgaD-D • Longer oligosaccharides cannot fit into the active site of BgaD-A • Structural differences explain the observed transgalactosylation specificities Hovorková et al. reveal structural differences between the β-galactosidase (BgaD) isoforms A and D and identify a flexible "Barrier domain 8" in BgaD-A that might obstruct longer oligosaccharide substrates. This study improves our understanding of the structural determinants governing β-galactosidase catalysis, which is of interest for tailored GOS production. [ABSTRACT FROM AUTHOR]

Binding site flexibility and dynamics strongly affect the ability of proteins to accommodate substrates and inhibitors. The significance of these properties is particularly pronounced for proteins that are inherently flexible, such as cytochrome P450 enzymes (CYPs). While the research on human CYPs provides detailed knowledge on both structural and functional level, such analyses are still lacking for their plant counterparts. This study aims to bridge this gap. We developed a novel computational pipeline consisting of two steps. Firstly, we use molecular dynamics (MD) simulations to capture the full conformational ensemble for a certain plant CYP. Subsequently, we developed and applied a comprehensive methodology to analyze a number of binding site properties—size, flexibility, shape, hydrophobicity, and accessibility—using the fpocket and mdpocket packages on MD-generated trajectories. The workflow was validated on human CYPs 1A2, 2A6, and 3A4, as their binding site characteristics are well known. Not only could we confirm known binding site properties, but we also identified and named previously unseen binding site channels for CYPs 1A2 and 2A6. The pipeline was then applied to plant CYPs, leading to the first categorization of 15 chosen plant CYPs based on their binding site's (dis)similarities. This study provides a foundation for the largely uncharted fields of plant CYP substrate specificity and facilitates a more precise understanding of their largely unknown specific biological functions. It offers new insights into the structural and functional dynamics of plant CYPs, which may facilitate a more accurate understanding of the fate of agrochemicals or the biotechnological design and exploitation of enzymes with specific functions. Additionally, it serves as a reference for future structural–functional analyses of CYP enzymes across various biological kingdoms. [ABSTRACT FROM AUTHOR]

The cytochrome P450s enzyme family metabolizes ∼80% of small molecule drugs. Variants in cytochrome P450s can substantially alter drug metabolism, leading to improper dosing and severe adverse drug reactions. Due to low sequence conservation, predicting variant effects across cytochrome P450s is challenging. Even closely related cytochrome P450s like CYP2C9 and CYP2C19, which share 92% amino acid sequence identity, display distinct phenotypic properties. Using variant abundance by massively parallel sequencing, we measured the steady-state protein abundance of 7,660 single amino acid variants in CYP2C19 expressed in cultured human cells. Our findings confirmed critical positions and structural features essential for cytochrome P450 function, and revealed how variants at conserved positions influence abundance. We jointly analyzed 4,670 variants whose abundance was measured in both CYP2C19 and CYP2C9, finding that the homologs have different variant abundances in substrate recognition sites within the hydrophobic core. We also measured the abundance of all single and some multiple wild type amino acid exchanges between CYP2C19 and CYP2C9. While most exchanges had no effect, substitutions in substrate recognition site 4 reduced abundance in CYP2C19. Double and triple mutants showed distinct interactions, highlighting a region that points to differing thermodynamic properties between the 2 homologs. These positions are known contributors to substrate specificity, suggesting an evolutionary tradeoff between stability and enzymatic function. Finally, we analyzed 368 previously unannotated human variants, finding that 43% had decreased abundance. By comparing variant effects between these homologs, we uncovered regions underlying their functional differences, advancing our understanding of this versatile family of enzymes. [ABSTRACT FROM AUTHOR]

AbstractDamaged 2’-deoxyribonucleotides cause mutations, cancer, cell death, and aging. The Escherichia coli Orf135 (NudG) protein catalyzes the hydrolysis of various 2’-deoxyribonucleotides including an oxidized form of dATP, 2-oxo-1,2-dihydro-2’-deoxyadenosine 5’-triphosphate (dAOTP, 2-hydroxy-2’-deoxyadenosine 5’-triphosphate). The best substrate is 5-methyl-2’-deoxycytidine 5’-triphosphate (dCmTP), and the protein prefers dCmTP over dAOTP by ∼200-fold in vitro. To make the enzyme specific for the mutagenic nucleotide dAOTP, a double mutant protein (E33A plus D118E) was designed and produced in E. coli. The purified mutant protein showed one order of magnitude higher dAOTP preference over dCmTP. The split protein based on this mutant may potentially be used to detect dAOTP in living cells. [ABSTRACT FROM AUTHOR]

Background: α‐l‐Fucose confers unique functions for fucose‐containing biomolecules such as human milk oligosaccharides. α‐l‐Fucosidases can serve as desirable tools in the application of fucosylated saccharides. Discovering novel α‐l‐fucosidases and elucidating their enzyme properties are always worthy tasks. Results: A GH95 family α‐l‐fucosidase named Afc95A_Wf was cloned from the genome of the marine bacterium Wenyingzhuangia fucanilytica and expressed in Escherichia coli. It exhibited maximum activity at 40 °C and pH 7.5. Afc95A_Wf defined a different substrate specificity among reported α‐l‐fucosidases, which was capable of hydrolyzing α‐fucoside in CNP‐fucose, Fucα1‐2Galβ1‐4Glc and Galβ1‐4(Fucα1‐3)Glc, and showed a preference for α1,2‐fucosidic linkage. It adopted Asp residue in the amino acid sequence at position 391, which was distinct from the previously acknowledged residue of Asn. The predicted tertiary structure and site‐directed mutagenesis revealed that Asp391 participates in the catalysis of Afc95A_Wf. The differences in the substrate specificity and catalytic site shed light on that Afc95A_Wf adopted a novel mechanism in catalysis. Conclusion: A GH95 family α‐l‐fucosidase (Afc95A_Wf) was cloned and expressed. It showed a cleavage preference for α1,2‐fucosidic linkage to α1,3‐fucosidic linkage. Afc95A_Wf demonstrated a different substrate specificity and a residue at an important catalytic site compared with known GH95 family proteins, which revealed the occurrence of diversity on catalytic mechanisms in the GH95 family. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The Zrt/Irt-like protein (ZIP) family consists of ubiquitously expressed divalent d-block metal transporters that play central roles in the uptake, secretion, excretion, and distribution of several essential and toxic metals in living organisms. The past few years has witnessed rapid progress in the molecular basis of these membrane transport proteins. In this critical review, we summarize the research progress at the molecular level of the ZIP family and discuss the future prospects. Furthermore, an evolutionary path for the unique ZIP fold and a new classification of the ZIP family are proposed based on the presented structural and sequence analyses. [ABSTRACT FROM AUTHOR]

Aggregation behavior provides bacteria protection from harsh environments and threats to survival. Two uncharacterized proteases, LapX and Lap, are important for Vibrio cholerae liquid-based aggregation. Here, we determined that LapX is a serine protease with a preference for cleavage after glutamate and glutamine residues in the P1 position, which processes a physiologically based peptide substrate with a catalytic efficiency of 180 ± 80 M-1s-1. The activity with a LapX substrate identified by a multiplex substrate profiling by mass spectrometry screen was 590 ± 20 M-1s-1. Lap shares high sequence identity with an aminopeptidase (termed VpAP) from Vibrio proteolyticus and contains an inhibitory bacterial prepeptidase C-terminal domain that, when eliminated, increases catalytic efficiency on leucine p-nitroanilide nearly four-fold from 5.4 ± 4.1 × 104 M-1s-1 to 20.3 ± 4.3 × 104 M-1s-1. We demonstrate that LapX processes Lap to its mature form and thus amplifies Lap activity. The increase is approximately eighteen-fold for full-length Lap (95.7 ± 5.6 × 104 M-1s-1) and six-fold for Lap lacking the prepeptidase C-terminal domain (11.3 ± 1.9 × 105 M-1s-1). In addition, substrate profiling reveals preferences for these two proteases that could inform in vivo function. Furthermore, purified LapX and Lap restore the timing of the V. cholerae aggregation program to a mutant lacking the lapX and lap genes. Both proteases must be present to restore WT timing, and thus they appear to act sequentially: LapX acts on Lap, and Lap acts on the substrate involved in aggregation.

DNA methylation provides an important epigenetic mechanism that critically regulates gene expression, genome imprinting, and retrotransposon silencing. In plants, DNA methylation is prevalent not only in a CG dinucleotide context but also in non-CG contexts, namely CHG and CHH (H = C, T, or A) methylation. It has been established that plant non-CG DNA methylation is highly context dependent, with the +1- and +2-flanking sequences enriched with A/T nucleotides. How DNA sequence, conformation, and dynamics influence non-CG methylation remains elusive. Here, we report structural and biochemical characterizations of the intrinsic substrate preference of DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), a plant DNA methyltransferase responsible for establishing all cytosine methylation and maintaining CHH methylation. Among nine CHH motifs, the DRM2 methyltransferase (MTase) domain shows marked substrate preference toward CWW (W = A or T) motifs, correlating well with their relative abundance in planta. Furthermore, we report the crystal structure of DRM2 MTase in complex with a DNA duplex containing a flexible TpA base step at the +1/+2-flanking sites of the target nucleotide. Comparative structural analysis of the DRM2-DNA complexes provides a mechanism by which flanking nucleotide composition impacts DRM2-mediated DNA methylation. Furthermore, the flexibility of the TpA step gives rise to two alternative DNA conformations, resulting in different interactions with DRM2 and consequently temperature-dependent shift of the substrate preference of DRM2. Together, this study provides insights into how the interplay between the conformational dynamics of DNA and temperature as an environmental factor contributes to the context-dependent CHH methylation by DRM2.