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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.

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.

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]

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]

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]

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]

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]

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]

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]

SUMMARY: Triterpenoids (C30‐isoprenoids) represent a major group of natural products with various physiological functions in plants. Triterpenoids and their derivatives have medicinal uses owing to diverse bioactivities. Arjuna (Terminalia arjuna) tree bark accumulates highly oxygenated β‐amyrin‐derived oleanane triterpenoids (e.g., arjunic acid, arjungenin, and arjunolic acid) with cardioprotective roles. However, biosynthetic routes and enzymes remain poorly understood. We mined the arjuna transcriptome and conducted cytochrome P450 monooxygenase (P450) assays using Saccharomyces cerevisiae and Nicotiana benthamiana to identify six P450s and two P450 reductases for oxidative modifications of oleanane triterpenoids. P450 assays using oleananes revealed a greater substrate promiscuity of C‐2α and C‐23 hydroxylases/oxidases than C‐28 oxidases. CYP716A233 and CYP716A432 catalyzed β‐amyrin/erythrodiol C‐28 oxidation to produce oleanolic acid. C‐2α hydroxylases (CYP716C88 and CYP716C89) converted oleanolic acid and hederagenin to maslinic acid and arjunolic acid. CYP716C89 also hydroxylated erythrodiol and oleanolic aldehyde. However, CYP714E107a and CYP714E107b catalyzed oleanolic acid/maslinic acid/arjunic acid, C‐23 hydroxylation to form hederagenin, arjunolic acid and arjungenin, and hederagenin C‐23 oxidation to produce gypsogenic acid, but at a lower rate than oleanolic acid C‐23 hydroxylation. Overall, P450 substrate selectivity suggested that C‐28 oxidation is the first P450‐catalyzed oxidative modification in the arjuna triterpenoid pathway. However, the pathway might branch thereafter through C‐2α/C‐23 hydroxylation of oleanolic acid. Taken together, these results provided new insights into substrate range of P450s and unraveled biosynthetic routes of triterpenoids in arjuna. Moreover, complete elucidation and reconstruction of arjunolic acid pathway in S. cerevisiae and N. benthamiana suggested the utility of arjuna P450s in heterologous production of cardioprotective compounds. Significance Statement: Arjuna tree bark contains bioactive triterpenoids that received considerable attention as cardioprotective compounds, but the biosynthetic enzymes remained largely unknown. In this work, transcriptome mining, metabolite analysis, and enzyme biochemical characterization led to identification of six cytochrome P450 monooxygenases (CYP716A233, CYP716A432, CYP716C88, CYP716C89, CYP714E107a, and CYP714E107b) and two cognate P450 reductases (TaCPR1 and TaCPR2) that catalyzed sequential C‐2α, C‐23, and C‐28 oxidations of oleanane scaffolds for the biosynthesis of arjunic acid, arjungenin, and arjunolic acid. [ABSTRACT FROM AUTHOR]

BsCotA laccase is a promising candidate for industrial application due to its excellent thermal stability. In this research, our objective was to enhance the catalytic efficiency of BsCotA by modifying the active site pocket. We utilized a strategy combining the diversity design of the active site pocket with molecular docking screening, which resulted in selecting five variants for characterization. All five variants proved functional, with four demonstrating improved turnover rates. The most effective variants exhibited a remarkable 7.7-fold increase in catalytic efficiency, evolved from 1.54 × 105 M−1 s−1 to 1.18 × 106 M−1 s−1, without any stability loss. To investigate the underlying molecular mechanisms, we conducted a comprehensive structural analysis of our variants. The analysis suggested that substituting Leu386 with aromatic residues could enhance BsCotA's ability to accommodate the 2,2′-azino-di-(3-ethylbenzothiazoline)-6-sulfonate (ABTS) substrate. However, the inclusion of charged residues, G323D and G417H, into the active site pocket reduced kcat. Ultimately, our research contributes to a deeper understanding of the role played by residues in the laccases' active site pocket, while successfully demonstrating a method to lift the catalytic efficiency of BsCotA. Key points: • Active site pocket design that enhanced BsCotA laccase efficiency • 7.7-fold improved in catalytic rate • All tested variants retain thermal stability [ABSTRACT FROM AUTHOR]

Phenylpropanoid sucrose esters are a large and important group of natural substances with significant therapeutic potential. This work describes a pilot study of the enzymatic hydroxycinnamoylation of sucrose and its derivatives which was carried out with the aim of obtaining precursors of natural phenylpropanoid sucrose esters, e.g., vanicoside B. In addition to sucrose, some chemically prepared sucrose acetonides and substituted 3′-O-cinnamates were subjected to enzymatic transesterification with vinyl esters of coumaric, ferulic and 3,4,5-trimethoxycinnamic acid. Commercial enzyme preparations of Lipozyme TL IM lipase and Pentopan 500 BG exhibiting feruloyl esterase activity were tested as biocatalysts in these reactions. The substrate specificity of the used biocatalysts for the donor and acceptor as well as the regioselectivity of the reactions were evaluated and discussed. Surprisingly, Lipozyme TL IM catalyzed the cinnamoylation of sucrose derivatives more to the 1′-OH and 4′-OH positions than to the 6′-OH when the 3′-OH was free and the 6-OH was blocked by isopropylidene. In this case, Pentopan reacted comparably to 1′-OH and 6′-OH positions. If sucrose 3′-O-coumarate was used as an acceptor, in the case of feruloylation with Lipozyme in CH3CN, 6-O-ferulate was the main product (63%). Pentopan feruloylated sucrose 3′-O-coumarate comparably well at the 6-OH and 6′-OH positions (77%). When a proton-donor solvent was used, migration of the 3′-O-cinnamoyl group from fructose to the 2-OH position of glucose was observed. The enzyme hydroxycinnamoylations studied can shorten the targeted syntheses of various phenylpropanoid sucrose esters. [ABSTRACT FROM AUTHOR]

Gluconobacter oxydans succinic semialdehyde reductase (GoxSSAR) and Acetobacter aceti glyoxylate reductase (AacGR) represent a novel class in the β-hydroxyacid dehydrogenases superfamily. Kinetic analyses revealed GoxSSAR's activity with both glyoxylate and succinic semialdehyde, while AacGR is glyoxylate specific. GoxSSAR K167A lost activity with succinic semialdehyde but retained some with glyoxylate, whereas AacGR K175A lost activity. These findings elucidate differences between these homologous enzymes. [ABSTRACT FROM AUTHOR]

The main protease from coronaviruses and the 3C protease from enteroviruses play a crucial role in processing viral polyproteins, making them attractive targets for the development of antiviral agents. In this study, we employed a combinatorial chemistry approach—HyCoSuL—to compare the substrate specificity profiles of the main and 3C proteases from alphacoronaviruses, betacoronaviruses, and enteroviruses. The obtained data demonstrate that coronavirus Mpros exhibit overlapping substrate specificity in all binding pockets, whereas the 3Cpro from enterovirus displays slightly different preferences toward natural and unnatural amino acids at the P4‐P2 positions. However, chemical tools such as substrates, inhibitors, and activity‐based probes developed for SARS‐CoV‐2 Mpro can be successfully applied to investigate the activity of the Mpro from other coronaviruses as well as the 3Cpro from enteroviruses. Our study provides a structural framework for the development of broad‐spectrum antiviral compounds. [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.

Human cytochrome P450 3A4 (CYP3A4) is a major drug-metabolizing enzyme that shows extreme substrate promiscuity. Moreover, its large and malleable active site can simultaneously accommodate several substrate molecules of the same or different nature, which may lead to cooperative binding and allosteric behavior. Due to difficulty of crystallization of CYP3A4-substrate complexes, it remains unknown how multiple substrates can arrange in the active site. We determined crystal structures of CYP3A4 bound to three and six molecules of caffeine, a psychoactive alkaloid serving as a substrate and modulator of CYP3A4. In the ternary complex, one caffeine binds to the active site suitably for C8-hydroxylation, most preferable for CYP3A4. In the senary complex, three caffeine molecules stack parallel to the heme with the proximal ligand poised for 3-N-demethylation. However, the caffeine stack forms extensive hydrophobic interactions that could preclude product dissociation and multiple turnovers. In both complexes, caffeine is also bound in the substrate channel and on the outer surface known as a peripheral site. At all sites, aromatic stacking with the caffeine ring(s) is likely a dominant interaction, while direct and water-mediated polar contacts provide additional stabilization for the substrate-bound complexes. Protein-ligand interactions via the active site R212, intrachannel T224, and peripheral F219 were experimentally confirmed, and the latter two residues were identified as important for caffeine association. Collectively, the structural, spectral, and mutagenesis data provide valuable insights on the ligand binding mechanism and help better understand how purine-based pharmaceuticals and other aromatic compounds could interact with CYP3A4 and mediate drug-drug interactions.

To achieve efficient degradation of crustacean raw materials and eco-preparation of N-acetyl-oligosaccharides, YS-54 strain screened from offshore soil in Qingdao was used for chitinase preparation in the present study. Chitinase was prepared through 60% ammonium sulfate precipitation and Sephadex G-100 gel filtration chromatography. Enzymatic properties such as optimal temperature, optimal pH, and substrate specificity were characterized. Mass spectrometry was used to determine the polymerization degree of enzymatic products. The 16S rRNA identification result showed that YS-54 strain belonged to the Aeromonas species. Chitinase from Aeromonas sp. YS-54 showed a specific activity of 23.44 U/mg. The molecular weights of chitinase were 63 and 75 kDa as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Chitinase was stable under the condition of 40 ℃ and pH5.0. The enzymatic activity was significantly activated by Ba2+, Co2+, Mg2+ and TritonX-100 (P

Abstract Bifidobacterium adolescentis gene BAD_1527 has previously been suggested to code for a β-xylosidase (Kobayashi et al., Mar Drugs 18:174, 2020). Our detailed investigation of the substrate specificity of the GH43_22 protein using a wide spectrum of natural and artificial substrates showed that the enzyme hydrolyzed neither linear xylooligosaccharides nor glucuronoxylan. Xylose was released only from the artificial 4-nitrophenyl β-D-xylopyranoside (1.58 mU/mg). The corresponding α-L-arabinofuranoside was by three orders of magnitude better substrate (2.17 U/mg). Arabinose was the only monosaccharide liberated from arabinoxylan and α-1,3- or α-1,2-singly arabinosylated xylooligosaccharides. Moreover, the enzyme efficiently debranched sugar beet arabinan and singly arabinosylated α-1,5-L-arabinooligosaccharides, although short linear α-1,5-L-arabinooligosaccharides were also slowly degraded. On the other hand, debranched arabinan, arabinogalactan as well as 2,3-doubly arabinosylated main chain residues of arabinan and arabinoxylan did not serve as substrates. Thus, the enzyme encoded by the BAD_1527 gene is a typical α-L-arabinofuranosidase of AXH-m specificity.

Abstract l-2-Keto-3-deoxyfuconate 4-dehydrogenase (l-KDFDH) catalyzes the NAD+-dependent oxidization of l-2-keto-3-deoxyfuconate (l-KDF) to l-2,4-diketo-3-deoxyfuconate (l-2,4-DKDF) in the non-phosphorylating l-fucose pathway from bacteria, and its substrate was previously considered to be the acyclic α-keto form of l-KDF. On the other hand, BDH2, a mammalian homolog with l-KDFDH, functions as a dehydrogenase for cis-4-hydroxy-l-proline (C4LHyp) with the cyclic structure. We found that l-KDFDH and BDH2 utilize C4LHyp and l-KDF, respectively. Therefore, to elucidate unique substrate specificity at the atomic level, we herein investigated for the first time the crystal structures of l-KDFDH from Herbaspirillum huttiense in the ligand-free, l-KDF and l-2,4-DKDF, d-KDP (d-2-keto-3-deoxypentonate; additional substrate), or l-2,4-DKDF and NADH bound forms. In complexed structures, l-KDF, l-2,4-DKDF, and d-KDP commonly bound as a α-furanosyl hemiketal. Furthermore, l-KDFDH showed no activity for l-KDF and d-KDP analogs without the C5 hydroxyl group, which form only the acyclic α-keto form. The C1 carboxyl and α-anomeric C2 hydroxyl groups and O5 oxygen atom of the substrate (and product) were specifically recognized by Arg148, Arg192, and Arg214. The side chain of Trp252 was important for hydrophobically recognizing the C6 methyl group of l-KDF. This is the first example showing the physiological role of the hemiketal of 2-keto-3-deoxysugar acid.

To achieve efficient degradation of crustacean raw materials and eco-preparation of N-acetyl-oligosaccharides, YS-54 strain screened from offshore soil in Qingdao was used for chitinase preparation in the present study. Chitinase was prepared through 60% ammonium sulfate precipitation and Sephadex G-100 gel filtration chromatography. Enzymatic properties such as optimal temperature, optimal pH, and substrate specificity were characterized. Mass spectrometry was used to determine the polymerization degree of enzymatic products. The 16S rRNA identification result showed that YS-54 strain belonged to the Aeromonas species. Chitinase from Aeromonas sp. YS-54 showed a specific activity of 23.44 U/mg. The molecular weights of chitinase were 63 and 75 kDa as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Chitinase was stable under the condition of 40 °C and pH5.0. The enzymatic activity was significantly activated by Ba2+, Co2+, Mg2+ and TritonX-100 (P<0.05), while inhibited by Fe3+, Cu2+ and SDS. The specific activity of chitinase toward α-chitin was 7.99 U/mg, 34.09% of that toward colloidal chitin. The polymerization degree of enzymatic hydrolysis products from colloidal chitin and α-chitin were 1~4 and 1~3, respectively. Chitinase from Aeromonas sp. YS-54 was stable in broad range of temperature and pH, meanwhile showed high catalytic efficiency toward α-chitin. The hydrolysis characteristics of chitinase from Aeromonas sp. YS-54 would provide technical support for the comprehensive utilization of crustacean waste. [ABSTRACT FROM AUTHOR]

Polyphosphate kinases (PPKs) are valuable biocatalysts for ATP cofactor regeneration as well as for the phosphorylation of non‐canonical nucleotides. The versatility of PPKs in the latter application is defined by their substrate scope. In this study, we investigate the substrate spectrum of the PPK2‐III enzyme from Meiothermus ruber (MrPPK) for the conversion of canonical and non‐canonical (deoxy)‐ribonucleotides. The enzyme shows high substrate promiscuity for purine and to minor degree pyrimidine substrates, with an overall preference for the native substrate AMP. With structure guided rational amino acid exchanges in the active site, we produced an MrPPK variant with improved activity for a broad variety of purine nucleotides. While the preference for AMP is lost, conversion of nucleotides without a 6‐amino function at the purine moiety is increased. This MrPPK variant is a versatile biocatalyst for the synthesis of non‐canonical nucleotides and could also be useful as a GTP cofactor regeneration system. [ABSTRACT FROM AUTHOR]

A type III polyketide synthase (SfuPKS2) from the edible seaweed Sargassum fusiforme was cloned and biochemically characterized. SfuPKS2 can only accept octanoyl-CoA (C8) and neither other long-chain fatty acyl-CoAs, nor non-fatty acyl-CoAs synthesized in this study as the substrate. In addition, in incubation with octanoyl-CoA, only a single product peak was observed, giving rise to the triketide α-pyrone derivative. To understand the substrate specificity of SfuPKS2, we constructed a structural model and performed docking analysis to reveal amino acid residues critical for enzyme-substrate binding. Putative key residues were then mutated experimentally and the impact on protein function was assessed by incubating the mutant with octanoyl-CoA. We found that key amino acids governing enzyme activity and specificity included Ser171, His236, Phe254, and Ile377. Mutations on these amino acids generally resulted in altered interactions with the substrate and thus the enzyme activity. In summary, our work provided mechanistic insight on how the selectivity toward the substrate is achieved in SfuPKS2. [ABSTRACT FROM AUTHOR]

The increasing number of uncharacterized proteins in the CAZy database highlights the importance of their functional characterization. Therefore, the substrate and positional specificity of 34 α-L-arabinofuranosidases classified into GH43, GH51, and GH62 families was determined on arabinoxylan, arabinan, and derived oligosaccharides (many enzyme–substrate combinations were examined for the first time) covering all possible kinds of arabinofuranosyl branches using TLC. Arabinoxylan was efficiently debranched by the majority of the tested proteins. Most of them showed AXH-m specificity, acting on 2- or 3-monoarabinosylated substrates, while AXH-d3 specificity (liberation of 3-linked arabinose solely from 2,3 doubly decorated substrates) was found mainly in the subfamily GH43_10, harbouring enzymes of both types. Several GH51 enzymes, however, released arabinose also from a xylooligosaccharide doubly arabinosylated at the non-reducing end. The AXH-m and AXH-d3 specificities correlated well with the dearabinosylation of arabinan and arabinooligosaccharides, which were debranched by all GH51 representatives and some GH43 and GH62 members. The GH51 and GH62 arabinan-debranching enzymes also hydrolyzed debranched arabinan, while within the GH43 family the linear arabinan-degrading ability was found only in the GH43_26 subfamily, comprising specific exo 1,5-α-L-arabinofuranosidases. This study demonstrates a first attempt in the systematic examination of a relationship between CAZy classification and substrate and positional specificities of various α-L-arabinofuranosidases. [ABSTRACT FROM AUTHOR]

The article investigates the impact of industrial microbial alkaline proteases from isolated bacillus strains. It discusses the applications of alkaline proteases in the food industry, detergent industry, leather industry, photographic industry, as well as in medicines, management of industrial and household waste, peptide synthesis and silk degumming.

Proper protein arginine methylation by protein arginine methyltransferase 1 (PRMT1) is critical for maintaining cellular health, while dysregulation is often associated with disease. How the activity of PRMT1 is regulated is therefore paramount, but is not clearly understood. Several studies have observed higher order oligomeric species of PRMT1, but it is unclear if these exist at physiological concentrations and there is confusion in the literature about how oligomerization affects activity. We therefore sought to determine which oligomeric species of PRMT1 are physiologically relevant, and quantitatively correlate activity with specific oligomer forms. Through quantitative western blotting, we determined that concentrations of PRMT1 available in a variety of human cell lines are in the sub‐micromolar to low micromolar range. Isothermal spectral shift binding data were modeled to a monomer/dimer/tetramer equilibrium with an EC50 for tetramer dissociation of ~20 nM. A combination of sedimentation velocity and Native polyacrylamide gel electrophoresis experiments directly confirmed that the major oligomeric species of PRMT1 at physiological concentrations would be dimers and tetramers. Surprisingly, the methyltransferase activity of a dimeric PRMT1 variant is similar to wild type, tetrameric PRMT1 with some purified substrates, but dimer and tetramer forms of PRMT1 show differences in catalytic efficiencies and substrate specificity for other substrates. Our results define an oligomerization paradigm for PRMT1, show that the biophysical characteristics of PRMT1 are poised to support a monomer/dimer/tetramer equilibrium in vivo, and suggest that the oligomeric state of PRMT1 could be used to regulate substrate specificity. [ABSTRACT FROM AUTHOR]

Enzymatic browning, catalysed by the enzyme polyphenol oxidase in fruit and vegetables, limits the efficient use of natural resources and promote food waste. Plums are a popular fruit with consumers around the world and are considered an important raw material in the food industry. Plums are very susceptible to enzymatic browning due to their high phenolic compound content and climacteric nature. The aim of this study is to purify the polyphenol oxidase enzyme from Sarali plum (Prunus domestica) and to determine its biochemical properties, kinetic parameters, pH and thermal stability and inhibition. In this study, polyphenol oxidase enzyme was purified 22.54-fold by affinity chromatography using Sepharose-4B-l-Tyr-p-amino benzoic acid affinity gel. The purity and molecular mass of the enzyme were determined by SDS-PAGE and non-denaturing PAGE (native PAGE). The molecular mass of the enzyme was estimated to be 72.44 kDa by SDS-PAGE. The enzyme was confirmed as PPO by native PAGE as a single band. Kinetic characterization studies were conducted for both catechol and 4-methyl catechol substrates. The optimal pH and temperature for both substrates were found to be 7.0 and 20 °C, respectively. The thermal stability of PPO was investigated, and it retained about 90% of its activity for 90 min at 4 °C. The determination of KM and Vmax was carried out using the Lineweaver–Burk plot. The substrate specificity (Vmax/KM) values for catechol and 4-methyl catechol were determined as 790.91 ± 37.34 and 492.06 ± 13.75 respectively. The enzyme exhibited the best activity towards catechol substrate. IC50, Ki constant and inhibition types were determined for various anti-browning agents on PPO enzyme. Ascorbic acid, l-cysteine, citric acid, salicylic acid and tartaric acid effectively inhibited PPO activity. [ABSTRACT FROM AUTHOR]

Bifidobacterium adolescentis gene BAD_1527 has previously been suggested to code for a β-xylosidase (Kobayashi et al., Mar Drugs 18:174, 2020). Our detailed investigation of the substrate specificity of the GH43_22 protein using a wide spectrum of natural and artificial substrates showed that the enzyme hydrolyzed neither linear xylooligosaccharides nor glucuronoxylan. Xylose was released only from the artificial 4-nitrophenyl β-D-xylopyranoside (1.58 mU/mg). The corresponding α-L-arabinofuranoside was by three orders of magnitude better substrate (2.17 U/mg). Arabinose was the only monosaccharide liberated from arabinoxylan and α-1,3- or α-1,2-singly arabinosylated xylooligosaccharides. Moreover, the enzyme efficiently debranched sugar beet arabinan and singly arabinosylated α-1,5-L-arabinooligosaccharides, although short linear α-1,5-L-arabinooligosaccharides were also slowly degraded. On the other hand, debranched arabinan, arabinogalactan as well as 2,3-doubly arabinosylated main chain residues of arabinan and arabinoxylan did not serve as substrates. Thus, the enzyme encoded by the BAD_1527 gene is a typical α-L-arabinofuranosidase of AXH-m specificity. Key points: BAD_1527 gene encodes a protein releasing xylose from NPX, but not natural substrates L-Arabinose is released from natural substrates, similarly to other GH43_22 members The enzyme debranches arabinoxylan, branched arabinan and derived oligosaccharides [ABSTRACT FROM AUTHOR]

Aminopeptidases with varied substrate specificities are involved in different crucial physiological processes of cellular homeostasis. They also have wide applications in food and pharma industries. Within the bacterial cell, broad specificity aminopeptidases primarily participate in the recycling of amino acids by degrading oligopeptides generated via primary proteolysis mediated by cellular ATP‐dependent proteases. However, in bacteria, a truly broad specificity enzyme, which can cleave off acidic, basic, Gly and hydrophobic amino acid residues, is extremely rare. Here, we report structure–function of a putative glycyl aminopeptidase (M61xc) from Xanthomonas campestris pv campestris (Xcc) belonging to the M61 peptidase family. The enzyme exhibits broad specificity and cleaves Ala, Leu, Asp, Glu, Met, Ser, Phe, Tyr, Gly, Arg, and Lys at the N terminus, optimally of peptides with a length of 3–7 amino acids. Further, we report the high‐resolution crystal structure of M61xc in the apo form (2.1 Å) and bestatin‐bound form (1.95 Å), detailing its catalytic and substrate preference mechanisms. Comparative analysis of enzyme activity in crude cell extracts from both wild‐type and m61xc‐knockout mutant strains of Xcc has elucidated the unique intracellular role of M61xc. This study suggests that M61xc is the exclusive enzyme in these bacteria that is responsible for liberating Asp/Glu residues from the N‐termini of peptides. Also, in view of its broad specificity and peptide degradation ability, it could be considered equivalent to M1 or other oligomeric peptidases from families like M17, M18, M42 or S9, who have an important auxiliary role in post‐proteasomal protein degradation in prokaryotes. [ABSTRACT FROM AUTHOR]

β‐ketoacyl ACP synthase I (KasA) has been considered as a promising drug target against Tuberculosis because it is known to play a pivotal role in the survival of Mycobacterium Tuberculosis, a causative agent of Tuberculosis. KasA catalyzes the reaction elongating only the acyl chain that is 16 carbon atoms in length or longer, but the molecular details of how KasA selectively recognizes only the substrate longer than a certain length still remain unknown. In the present study, this challenging subject is addressed, and to this end, molecular dynamics (MD) simulations and free energy calculations for actual substrate binding process are carried out. The results illustrate that the substrate specificity of KasA is highly linked to its cooperativity and this cooperativity is realized through the activation of catalytic residues. Through these results, the mechanistic details of how KasA can be selectively activated only by the substrate with a proper length are suggested. [ABSTRACT FROM AUTHOR]

There is mounting evidence that reproductively isolated, but morphologically weakly differentiated species (so-called cryptic species) represent a substantial part of biological diversity, especially in bryophytes. We assessed the evolutionary history and ecological differentiation of a species pair, Dicranum brevifolium and D. septentrionale, which have overlapping ranges in the Holarctic. Despite their morphological similarity, we found similar genetic differentiation as between morphologically well-differentiated Dicranum species. Moreover, we detected gene tree discordance between plastid and nuclear markers, but neither of the two datasets resolved the two as sister species. The signal in trnL–trnF better reflects the morphological and ecological affinities and indicates a close relationship while ITS sequence data resolved the two taxa as phylogenetically distantly related. The discordance is probably unrelated to the ecological differentiation of D. septentrionale to colonise subneutral to alkaline substrates (vs. acidic in D. brevifolium), because this ability is rare in the genus and shared with D. acutifolium. This taxon is the closest relative of D. septentrionale according to the trnL–trnF data and does not share the discordance in ITS. We furthermore demonstrate that beside D. acutifolium, both D. septentrionale and D. brevifolium occur in the Alps but D. brevifolium is most likely rarer. Based on morphological analyses including factor analysis for mixed data of 45 traits we suggest treating the latter two as near-cryptic species and we recommend verifying morphological determinations molecularly. [ABSTRACT FROM AUTHOR]

Alcohol oxidase (AOX) oxidizes alcohols to produce carbonyl compounds and peroxides. Its promoter (PAOX1) is widely used in methylotrophic yeasts. A promising yeast expression system (Pichia sp. strain SO) was developed for bacterial lipase expression regulated by PAOX1 of Komagataella phaffii (previously known as Pichia pastoris). However, its unidentified AOX gene and the protein structure have deterred the search for the best inducer. This study was aimed to identify the yeast species and determine the best inducer for PAOX1 upregulation using in silico AOX protein analysis. Morphological (scanning and transmission electron microscopies) and carbon assimilation analyses confirmed isolate SO as Meyerozyma guilliermondii (previously known as Pichia guilliiermondii). Using Hidden-Markov model and degenerate PCR, the LCAO gene (2091 bp) was discovered in M. guilliermondii strain SO. The enzyme, MgFAO1 shared 14% similarity to K. phaffii AOX1 protein (KpAOX1). Molecular docking of MgFAO1 three-dimensional structure predicted using AlphaFold2 showed its preference toward long-chain 1-dodecanol as the substrate unlike KpAOX1 (short-chain methanol). While the alcohol-binding pocket in MgFAO1 was more hydrophobic compared to KpAOX1, 1-dodecanol could be a better inducer for protein expression in M. guilliermondii strain SO. Thus, in silico pipeline employed in this study can help identify homologous proteins in other expression hosts and their preferred substrates for promoter upregulation. However, the computational analyses were merely predictions and further wet-lab validation is required. Yet, this strategy allows cost-efficient screening of potential inducers for microbe-based protein production in the industries, reducing the production cost and offering cheaper options for consumers. [ABSTRACT FROM AUTHOR]

Summary: Unlike 'white rot' (WR) wood‐decomposing fungi that remove lignin to access cellulosic sugars, 'brown rot' (BR) fungi selectively extract sugars and leave lignin behind. The relative frequency and distribution of these fungal types (decay modes) have not been thoroughly assessed at a global scale; thus, the fate of one‐third of Earth's aboveground carbon, wood lignin, remains unclear.Using c. 1.5 million fungal sporocarp and c. 30 million tree records from publicly accessible databases, we mapped and compared decay mode and tree type (conifer vs angiosperm) distributions. Additionally, we mined fungal record metadata to assess substrate specificity per decay mode.The global average for BR fungi proportion (BR/(BR + WR records)) was 13% and geographic variation was positively correlated (R2 = 0.45) with conifer trees proportion (conifer/(conifer + angiosperm records)). Most BR species (61%) were conifer, rather than angiosperm (22%), specialists. The reverse was true for WR (conifer: 19%; angiosperm: 62%). Global BR proportion patterns were predicted with greater accuracy using the relative distributions of individual tree species (R2 = 0.82), rather than tree type.Fungal decay mode distributions can be explained by tree type and, more importantly, tree species distributions, which our data suggest is due to strong substrate specificities. [ABSTRACT FROM AUTHOR]

Summary: Norspermine (Nspm), one of the uncommon polyamines (PAs), was detected in bryophytes and lycophytes; therefore, the aminopropyltransferases involved in the synthesis of Nspm were investigated.The enzymatic activity was evaluated by the transient high expression of various aminopropyltransferase genes in Nicotiana benthamiana, followed by quantification of PA distribution in the leaves using gas chromatography‐mass spectrometry.The bryophyte orthologues of ACL5, which is known to synthesise thermospermine (Tspm) in flowering plants, were found to have strong Nspm synthesis activity. In addition, two ACL5 orthologous with different substrate specificities were conserved in Selaginella moellendorffii, one of which was involved in Tspm synthesis and the other in Nspm synthesis. Therefore, further detailed analysis using these two factors revealed that the β‐hairpin structural region consisting of β‐strands 1 and 2 at the N‐terminus of ACL5 is involved in substrate specificity.Through functional analysis of a total of 40 ACL5 genes in 33 organisms, including algae, it was shown that ACL5 has changed its substrate specificity several times during plant evolution and diversification. Furthermore, it was strongly suggested that ACL5 acquired strict Tspm synthesis activity during the emergence of vascular plants, especially through major changes around the β‐hairpin structural region. [ABSTRACT FROM AUTHOR]

Human transglutaminase 1 (TG1) modulates skin development, while its involvement in diseases remains poorly understood, necessitating comprehensive exploration of its substrate interactions. To study the substrate profile of TG1, an in vitro selection system based on cDNA display technology was used to screen two peptide libraries with mutations at varying distance from the reactive glutamine. Next-generation sequencing and bioinformatics analysis of the selected DNA pools revealed a detailed TG1 substrate profile, indicating preferred and non-preferred amino acid sequences. The peptide sequence, AEQHKLPSKWPF, was identified showing high reactivity and specificity to TG1. The position weight matrix calculated from the per amino acid enrichment factors was employed to search human proteins using an in-house algorithm, revealing six known TG1 substrate proteins with high scores, alongside a list of candidate substrates currently under investigation. Our findings are expected to assist in future medical diagnoses and development of treatments for skin disorders. [ABSTRACT FROM AUTHOR]

Main conclusion: Overexpression of Artemisia annua jasmonic acid carboxyl methyltransferase (AaJMT) leads to enhanced artemisinin content in Artemisia annua. Artemisinin-based combination therapies remain the sole deterrent against deadly disease malaria and Artemisia annua remains the only natural producer of artemisinin. In this study, the 1101 bp gene S-adenosyl-l-methionine (SAM): Artemisia annua jasmonic acid carboxyl methyltransferase (AaJMT), was characterised from A. annua, which converts jasmonic acid (JA) to methyl jasmonate (MeJA). From phylogenetic analysis, we confirmed that AaJMT shares a common ancestor with Arabidopsis thaliana, Eutrema japonica and has a close homology with JMT of Camellia sinensis. Further, the Clustal Omega depicted that the conserved motif I, motif III and motif SSSS (serine) required to bind SAM and JA, respectively, are present in AaJMT. The relative expression of AaJMT was induced by wounding, MeJA and salicylic acid (SA) treatments. Additionally, we found that the recombinant AaJMT protein catalyses the synthesis of MeJA from JA with a Km value of 37.16 µM. Moreover, site-directed mutagenesis of serine-151 in motif SSSS to tyrosine, asparagine-10 to threonine and glutamine-25 to histidine abolished the enzyme activity of AaJMT, thus indicating their determining role in JA substrate binding. The GC–MS analysis validated that mutant proteins of AaJMT were unable to convert JA into MeJA. Finally, the artemisinin biosynthetic and trichome developmental genes were upregulated in AaJMT overexpression transgenic lines, which in turn increased the artemisinin content. [ABSTRACT FROM AUTHOR]

Human acyl protein thioesterases (APTs) catalyze the depalmitoylation of S‐acylated proteins attached to the plasma membrane, facilitating reversible cycles of membrane anchoring and detachment. We previously showed that a bacterial APT homologue, FTT258 from the gram‐negative pathogen Francisella tularensis, exists in equilibrium between a closed and open state based on the structural dynamics of a flexible loop overlapping its active site. Although the structural dynamics of this loop are not conserved in human APTs, the amino acid sequence of this loop is highly conserved, indicating essential but divergent functions for this loop in human APTs. Herein, we investigated the role of this loop in regulating the catalytic activity, ligand binding, and protein folding of human APT1, a depalmitoylase connected with cancer, immune, and neurological signaling. Using a combination of substitutional analysis with kinetic, structural, and biophysical characterization, we show that even in its divergent structural location in human APT1 that this loop still regulates the catalytic activity of APT1 through contributions to ligand binding and substrate positioning. We confirmed previously known roles for multiple residues (Phe72 and Ile74) in substrate binding and catalysis while adding new roles in substrate selectivity (Pro69), in catalytic stabilization (Asp73 and Ile75), and in transitioning between the membrane binding β‐tongue and substrate‐binding loops (Trp71). Even conservative substitution of this tryptophan (Trp71) fulcrum led to complete loss of catalytic activity, a 13°C decrease in total protein stability, and drastic drops in ligand affinity, indicating that the combination of the size, shape, and aromaticity of Trp71 are essential to the proper structure of APT1. Mixing buried hydrophobic surface area with contributions to an exposed secondary surface pocket, Trp71 represents a previously unidentified class of essential tryptophans within α/β hydrolase structure and a potential allosteric binding site within human APTs. [ABSTRACT FROM AUTHOR]

An increased understanding of how the acceptor site in Gcn5-related N-acetyltransferase (GNAT) enzymes recognizes various substrates provides important clues for GNAT functional annotation and their use as chemical tools. In this study, we explored how the PA3944 enzyme from Pseudomonas aeruginosa recognizes three different acceptor substrates, including aspartame, NANMO, and polymyxin B, and identified acceptor residues that are critical for substrate specificity. To achieve this, we performed a series of molecular docking simulations and tested methods to identify acceptor substrate binding modes that are catalytically relevant. We found that traditional selection of best docking poses by lowest S scores did not reveal acceptor substrate binding modes that were generally close enough to the donor for productive acetylation. Instead, sorting poses based on distance between the acceptor amine nitrogen atom and donor carbonyl carbon atom placed these acceptor substrates near residues that contribute to substrate specificity and catalysis. To assess whether these residues are indeed contributors to substrate specificity, we mutated seven amino acid residues to alanine and determined their kinetic parameters. We identified several residues that improved the apparent affinity and catalytic efficiency of PA3944, especially for NANMO and/or polymyxin B. Additionally, one mutant (R106A) exhibited substrate inhibition toward NANMO, and we propose scenarios for the cause of this inhibition based on additional substrate docking studies with R106A. Ultimately, we propose that this residue is a key gatekeeper between the acceptor and donor sites by restricting and orienting the acceptor substrate within the acceptor site.

The biological and pathological functions of cathepsin B occur in acidic lysosomes and at the neutral pH of cytosol, nuclei, and extracellular locations. Importantly, cathepsin B displays different substrate cleavage properties at acidic pH compared to neutral pH conditions. It is, therefore, desirable to develop specific substrates for cathepsin B that measure its activity over broad pH ranges. Current substrates used to monitor cathepsin B activity consist of Z-Phe-Arg-AMC and Z-Arg-Arg-AMC, but they lack specificity since they are cleaved by other cysteine cathepsins. Furthermore, Z-Arg-Arg-AMC monitors cathepsin B activity at neutral pH and displays minimal activity at acidic pH. Therefore, the purpose of this study was to design and validate specific fluorogenic peptide substrates that can monitor cathepsin B activity over a broad pH range from acidic to neutral pH conditions. In-depth cleavage properties of cathepsin B were compared to those of the cysteine cathepsins K, L, S, V, and X via multiplex substrate profiling by mass spectrometry at pH 4.6 and pH 7.2. Analysis of the cleavage preferences predicted the tripeptide Z-Nle-Lys-Arg-AMC as a preferred substrate for cathepsin B. Significantly, Z-Nle-Lys-Arg-AMC displayed the advantageous properties of measuring high cathepsin B specific activity over acidic to neutral pHs and was specifically cleaved by cathepsin B over the other cysteine cathepsins. Z-Nle-Lys-Arg-AMC specifically monitored cathepsin B activity in neuronal and glial cells which were consistent with relative abundances of cathepsin B protein. These findings validate Z-Nle-Lys-Arg-AMC as a novel substrate that specifically monitors cathepsin B activity over a broad pH range.

FAM86A is a class I lysine methyltransferase (KMT) that generates trimethylation on the eukaryotic translation elongation factor 2 (EEF2) at Lys525. Publicly available data from The Cancer Dependency Map project indicate high dependence of hundreds of human cancer cell lines on FAM86A expression. This classifies FAM86A among numerous other KMTs as potential targets for future anticancer therapies. However, selective inhibition of KMTs by small molecules can be challenging due to high conservation within the S-adenosyl methionine (SAM) cofactor binding domain among KMT subfamilies. Therefore, understanding the unique interactions within each KMT-substrate pair can facilitate developing highly specific inhibitors. The FAM86A gene encodes an N-terminal FAM86 domain of unknown function in addition to its C-terminal methyltransferase domain. Here, we used a combination of X-ray crystallography, the AlphaFold algorithms, and experimental biochemistry to identify an essential role of the FAM86 domain in mediating EEF2 methylation by FAM86A. To facilitate our studies, we also generated a selective EEF2K525 methyl antibody. Overall, this is the first report of a biological function for the FAM86 structural domain in any species and an example of a noncatalytic domain participating in protein lysine methylation. The interaction between the FAM86 domain and EEF2 provides a new strategy for developing a specific FAM86A small molecule inhibitor, and our results provide an example in which modeling a protein-protein interaction with AlphaFold expedites experimental biology.

Orchestrated action of peptidoglycan (PG) synthetases and hydrolases is vital for bacterial growth and viability. Although the function of several PG synthetases and hydrolases is well understood, the function, regulation, and mechanism of action of PG hydrolases characterised as lysostaphin-like endopeptidases have remained elusive. Many of these M23 family members can hydrolyse glycyl-glycine peptide bonds and show lytic activity against Staphylococcus aureus whose PG contains a pentaglycine bridge, but their exact substrate specificity and hydrolysed bonds are still vaguely determined. In this work, we have employed NMR spectroscopy to study both the substrate specificity and the bond cleavage of the bactericide lysostaphin and the S. aureus PG hydrolase LytM. Yet, we provide substrate-level evidence for the functional role of these enzymes. Indeed, our results show that the substrate specificities of these structurally highly homologous enzymes are similar, but unlike observed earlier both LytM and lysostaphin prefer the D-Ala-Gly cross-linked part of mature peptidoglycan. However, we show that while lysostaphin is genuinely a glycyl-glycine hydrolase, LytM can also act as a D-alanyl-glycine endopeptidase.

Ethylamine (EA), the precursor of theanine biosynthesis, is synthesized from alanine decarboxylation by alanine decarboxylase (AlaDC) in tea plants. AlaDC evolves from serine decarboxylase (SerDC) through neofunctionalization and has lower catalytic activity. However, lacking structure information hinders the understanding of the evolution of substrate specificity and catalytic activity. In this study, we solved the X-ray crystal structures of AlaDC from Camellia sinensis (CsAlaDC) and SerDC from Arabidopsis thaliana (AtSerDC). Tyr341 of AtSerDC or the corresponding Tyr336 of CsAlaDC is essential for their enzymatic activity. Tyr111 of AtSerDC and the corresponding Phe106 of CsAlaDC determine their substrate specificity. Both CsAlaDC and AtSerDC have a distinctive zinc finger and have not been identified in any other Group II PLP-dependent amino acid decarboxylases. Based on the structural comparisons, we conducted a mutation screen of CsAlaDC. The results indicated that the mutation of L110F or P114A in the CsAlaDC dimerization interface significantly improved the catalytic activity by 110% and 59%, respectively. Combining a double mutant of CsAlaDCL110F/P114A with theanine synthetase increased theanine production 672% in an in vitro system. This study provides the structural basis for the substrate selectivity and catalytic activity of CsAlaDC and AtSerDC and provides a route to more efficient biosynthesis of theanine.

Modular polyketide synthases (PKSs) are polymerases that employ α-carboxyacyl-CoAs as extender substrates. This enzyme family contains several catalytic modules, where each module is responsible for a single round of polyketide chain extension. Although PKS modules typically use malonyl-CoA or methylmalonyl-CoA for chain elongation, many other malonyl-CoA analogues are used to diversify polyketide structures in nature. Previously, we developed a method to alter an extension substrate of a given module by exchanging an acyltransferase (AT) domain while maintaining protein folding. Here, we report in vitro polyketide biosynthesis by 13 PKSs (the wild-type PKS and 12 AT-exchanged PKSs with unusual ATs) and 14 extender substrates. Our ∼200 in vitro reactions resulted in 13 structurally different polyketides, including several polyketides that have not been reported. In some cases, AT-exchanged PKSs produced target polyketides by >100-fold compared to the wild-type PKS. These data also indicate that most unusual AT domains do not incorporate malonyl-CoA and methylmalonyl-CoA but incorporate various rare extender substrates that are equal to in size or slightly larger than natural substrates. We developed a computational workflow to predict the approximate AT substrate range based on active site volumes to support the selection of ATs. These results greatly enhance our understanding of rare AT domains and demonstrate the benefit of using the proposed PKS engineering strategy to produce novel chemicals in vitro.

Our sense of smell enables us to navigate a vast space of chemically diverse odour molecules. This task is accomplished by the combinatorial activation of approximately 400 odorant G protein-coupled receptors encoded in the human genome1-3. How odorants are recognized by odorant receptors remains unclear. Here we provide mechanistic insight into how an odorant binds to a human odorant receptor. Using cryo-electron microscopy, we determined the structure of the active human odorant receptor OR51E2 bound to the fatty acid propionate. Propionate is bound within an occluded pocket in OR51E2 and makes specific contacts critical to receptor activation. Mutation of the odorant-binding pocket in OR51E2 alters the recognition spectrum for fatty acids of varying chain length, suggesting that odorant selectivity is controlled by tight packing interactions between an odorant and an odorant receptor. Molecular dynamics simulations demonstrate that propionate-induced conformational changes in extracellular loop 3 activate OR51E2. Together, our studies provide a high-resolution view of chemical recognition of an odorant by a vertebrate odorant receptor, providing insight into how this large family of G protein-coupled receptors enables our olfactory sense.

Aminotransferases (ATs) catalyze pyridoxal 5'-phosphate-dependent transamination reactions between amino donor and keto acceptor substrates and play central roles in nitrogen metabolism of all organisms. ATs are involved in the biosynthesis and degradation of both proteinogenic and nonproteinogenic amino acids and also carry out a wide variety of functions in photorespiration, detoxification, and secondary metabolism. Despite the importance of ATs, their functionality is poorly understood as only a small fraction of putative ATs, predicted from DNA sequences, are associated with experimental data. Even for characterized ATs, the full spectrum of substrate specificity, among many potential substrates, has not been explored in most cases. This is largely due to the lack of suitable high-throughput assays that can screen for AT activity and specificity at scale. Here we present a new high-throughput platform for screening AT activity using bioconjugate chemistry and mass spectrometry imaging-based analysis. Detection of AT reaction products is achieved by forming an oxime linkage between the ketone groups of transaminated amino donors and a probe molecule that facilitates mass spectrometry-based analysis using nanostructure-initiator mass spectrometry or MALDI-mass spectrometry. As a proof-of-principle, we applied the newly established method and found that a previously uncharacterized Arabidopsis thaliana tryptophan AT-related protein 1 is a highly promiscuous enzyme that can utilize 13 amino acid donors and three keto acid acceptors. These results demonstrate that this oxime-mass spectrometry imaging AT assay enables high-throughput discovery and comprehensive characterization of AT enzymes, leading to an accurate understanding of the nitrogen metabolic network.