Crystal structure of activating sulfotransferase SgdX2 involved in biosynthesis of secondary metabolite sungeidine.

Microorganisms synthesize a plethora of complex secondary metabolites, many of which are beneficial to human health, such as anticancer agents and antibiotics. Among these, the Sungeidines are a distinct class of secondary metabolites known for their bulky and intricate structures. They are produced by a specific biosynthetic gene cluster within the genome of the soil-dwelling actinomycete Micromonospora sp. MD118. A notable enzyme in the Sungeidine biosynthetic pathway is the activating sulfotransferase SgdX2. In this pathway, SgdX2 mediates a key sulfation step, after which the product undergoes spontaneous dehydration to yield a Sungeidine compound. To delineate the structural basis for SgdX2's substrate recognition and catalytic action, we have determined the crystal structure of SgdX2 in complex with its sulfate donor product, 3'-phosphoadenosine 5'-phosphate (PAP), at a resolution of 1.6 Å. Although SgdX2 presents a compact overall structure, its core elements are conserved among other activating sulfotransferases. Our structural analysis reveals a unique substrate-binding pocket that accommodates bulky, complex substrates, suggesting a specialized adaptation for Sungeidine synthesis. Moreover, we have constructed a substrate docking model that provides insights into the molecular interactions between SgdX2 and Sungeidine F, enhancing our understanding of the enzyme's specificity and catalytic mechanism. The model supports a general acid-base catalysis mechanism, akin to other sulfotransferases, and underscores the minor role of disordered regions in substrate recognition. This integrative study of crystallography and computational modeling advances our knowledge of microbial secondary metabolite biosynthesis and may facilitate the development of novel biotechnological applications.

A new type of sulfation reaction: C-sulfonation for α,ß-unsaturated carbonyl groups by a novel sulfotransferase SULT7A1.

Cytosolic sulfotransferases (SULTs) are cytosolic enzymes that catalyze the transfer of sulfonate group to key endogenous compounds, altering the physiological functions of their substrates. SULT enzymes catalyze the O-sulfonation of hydroxy groups or N-sulfonation of amino groups of substrate compounds. In this study, we report the discovery of C-sulfonation of α,ß-unsaturated carbonyl groups mediated by a new SULT enzyme, SULT7A1, and human SULT1C4. Enzymatic assays revealed that SULT7A1 is capable of transferring the sulfonate group from 3'-phosphoadenosine 5'-phosphosulfate to the α-carbon of α,ß-unsaturated carbonyl-containing compounds, including cyclopentenone prostaglandins as representative endogenous substrates. Structural analyses of SULT7A1 suggest that the C-sulfonation reaction is catalyzed by a novel mechanism mediated by His and Cys residues in the active site. Ligand-activity assays demonstrated that sulfonated 15-deoxy prostaglandin J2 exhibits antagonist activity against the prostaglandin receptor EP2 and the prostacyclin receptor IP. Modification of α,ß-unsaturated carbonyl groups via the new prostaglandin-sulfonating enzyme, SULT7A1, may regulate the physiological function of prostaglandins in the gut. Discovery of C-sulfonation of α,ß-unsaturated carbonyl groups will broaden the spectrum of potential substrates and physiological functions of SULTs.

Crystal structure of tick tyrosylprotein sulfotransferase reveals the activation mechanism of the tick anticoagulant protein madanin.

Ticks pose a substantial public health risk as they transmit various pathogens. This concern is related to the adept blood-sucking strategy of ticks, underscored by the action of the anticoagulant, madanin, which is known to exhibit an approximately 1000-fold increase in anticoagulant activity following sulfation of its two tyrosine residues, Tyr51 and Tyr54. Despite this knowledge, the molecular mechanism underlying sulfation by tick tyrosylprotein sulfotransferase (TPST) remains unclear. In this study, we successfully prepared tick TPST as a soluble recombinant enzyme. We clarified the method by which this enzyme proficiently sulfates tyrosine residues in madanin. Biochemical analysis using a substrate peptide based on madanin and tick TPST, along with the analysis of the crystal structure of the complex and docking simulations, revealed a sequential sulfation process. Initial sulfation at the Tyr51 site augments binding, thereby facilitating efficient sulfation at Tyr54. Beyond direct biochemical implications, these findings considerably improve our understanding of tick blood-sucking strategies. Furthermore, combined with the utility of modified tick TPST, our findings may lead to the development of novel anticoagulants, promising avenues for thrombotic disease intervention and advancements in the field of public health.

Crystal structure of mango α1,3/α1,4-fucosyltransferase elucidates unique elements that regulate Lewis A-dominant oligosaccharide assembly.

In various organisms, α1,3/α1,4-fucosyltransferases (CAZy GT10 family enzymes) mediate the assembly of type I (Galß1,3GlcNAc) and/or type II (Galß1,4GlcNAc)-based Lewis structures that are widely distributed in glycoconjugates. Unlike enzymes of other species, plant orthologues show little fucosyltransferase activity for type II-based glycans and predominantly catalyze the assembly of the Lewis A structure [Galß1,3(Fucα1,4)GlcNAc] on the type I disaccharide unit of their substrates. However, the structural basis underlying this unique substrate selectivity remains elusive. In this study, we investigated the structure-function relationship of MiFUT13A, a mango α1,3/α1,4-fucosyltransferase. The prepared MiFUT13A displayed distinct α1,4-fucosyltransferase activity. Consistent with the enzymatic properties of this molecule, X-ray crystallography revealed that this enzyme has a typical GT-B fold-type structure containing a set of residues that are responsible for its SN2-like catalysis. Site-directed mutagenesis and molecular docking analyses proposed a rational binding mechanism for type I oligosaccharides. Within the catalytic cleft, the pocket surrounding Trp121 serves as a binding site, anchoring the non-reducing terminal ß1,3-galactose that belongs to the type I disaccharide unit. Furthermore, Glu177 was postulated to function as a general base catalyst through its interaction with the 4-hydroxy group of the acceptor N-acetylglucosamine residue. Adjacent residues, specifically Thr120, Thr157 and Asp175 were speculated to assist in binding of the reducing terminal residues. Intriguingly, these structural elements were not fully conserved in mammalian orthologue which also shows predominant α1,4-fucosyltransferase activity. In conclusion, we have proposed that MiFUT13A generates the Lewis A structure on type I glycans through a distinct mechanism, divergent from that of mammalian enzymes.

Disassembly and reassembly of the non-conventional thermophilic C-phycocyanin.

C-phycocyanin (CPC), which contains open-chain tetrapyrroles, is a major light-harvesting red-fluorescent protein with an important role in aquatic photosynthesis. Recently, we reported a non-conventional CPC from Thermoleptolyngbya sp. O-77 (CPCO77) that contains two different structures, i.e., a hexameric structure and a non-conventional octameric structure. However, the assembly and disassembly mechanisms of the non-conventional octameric form of CPC remain unclear. To understand this assembly mechanism, we performed an in vitro experiment to study the disassembly and reassembly behaviors of CPC using isolated CPC subunits. The dissociation of the CPCO77 subunit was performed using a Phenyl-Sepharose column in 20 mM potassium phosphate buffer (pH 6.0) containing 7.0 M urea. For the first time, crystals of isolated CPC subunits were obtained and analyzed after separation. After the removal of urea from the purified α and ß subunits, we performed an in vitro reassembly experiment for CPC and analyzed the reconstructed CPC using spectrophotometric and X-ray crystal structure analyses. The crystal structure of the reassembled CPC was nearly identical to that of the original CPCO77. The findings of this study indicate that the octameric CPCO77 is a naturally occurring form in the thermophilic cyanobacterium Thermoleptolyngbya sp. O-77.

Crystal structure of Arabidopsis thaliana sulfotransferase SOT16 involved in glucosinolate biosynthesis.

Glucosinolates (GSLs), a class of secondary metabolites found in Brassicaceae plants, play important roles in plant defense and contribute distinct flavors and aromas when used as food ingredients. Following tissue damage, GSLs undergo enzymatic hydrolysis to release bioactive volatile compounds. Understanding GSL biosynthesis and enzyme involvement is crucial for improving crop quality and advancing agriculture. Plant sulfotransferases (SOTs) play a key role in the final step of GSL biosynthesis by transferring sulfate groups to the precursor molecules. In the present study, we investigated the enzymatic reaction mechanism and broad substrate specificity of Arabidopsis thaliana sulfotransferase AtSOT16, which is involved in GSL biosynthesis, using crystal structure analysis. Our analysis revealed the specific catalytic residues involved in the sulfate transfer reaction and supported the hypothesis of a concerted acid-base catalytic mechanism. Furthermore, the docking models showed a strong correlation between the substrates with high predicted binding affinities and those experimentally reported to exhibit high activity. These findings provide valuable insights into the enzymatic reaction mechanisms and substrate specificity of GSL biosynthesis. The information obtained in this study may contribute to the development of novel strategies for manipulating GSL synthesis pathways in Brassica plants and has potential agricultural applications.

Crystal structure of adenosine 5'-phosphosulfate kinase isolated from Archaeoglobus fulgidus.

The 3'-phosphoadenosine-5'-phosphosulfate (PAPS) molecule is essential during enzyme-catalyzed sulfation reactions as a sulfate donor and is an intermediate in the reduction of sulfate to sulfite in the sulfur assimilation pathway. PAPS is produced through a two-step reaction involving ATP sulfurylase and adenosine 5'-phosphosulfate (APS) kinase enzymes/domains. However, archaeal APS kinases have not yet been characterized and their mechanism of action remains unclear. Here, we first structurally characterized APS kinase from the hyperthermophilic archaeon Archaeoglobus fulgidus, (AfAPSK). We demonstrated the PAPS production activity of AfAPSK at the optimal growth temperature (83 °C). Furthermore, we determined the two crystal structures of AfAPSK: ADP complex and ATP analog adenylyl-imidodiphosphate (AMP-PNP)/Mg2+/APS complex. Structural and complementary mutational analyses revealed the catalytic and substrate recognition mechanisms of AfAPSK. This study also hints at the molecular basis behind the thermal stability of AfAPSK.

Construction of a Versatile, Programmable RNA-Binding Protein Using Designer PPR Proteins and Its Application for Splicing Control in Mammalian Cells.

RNAs play many essential roles in gene expression and are involved in various human diseases. Although genome editing technologies have been established, the engineering of sequence-specific RNA-binding proteins that manipulate particular cellular RNA molecules is immature, in contrast to nucleotide-based RNA manipulation technology, such as siRNA- and RNA-targeting CRISPR/Cas. Here, we demonstrate a versatile RNA manipulation technology using pentatricopeptide-repeat (PPR)-motif-containing proteins. First, we developed a rapid construction and evaluation method for PPR-based designer sequence-specific RNA-binding proteins. This system has enabled the steady construction of dozens of functional designer PPR proteins targeting long 18 nt RNA, which targets a single specific RNA in the mammalian transcriptome. Furthermore, the cellular functionality of the designer PPR proteins was first demonstrated by the control of alternative splicing of either a reporter gene or an endogenous CHK1 mRNA. Our results present a versatile protein-based RNA manipulation technology using PPR proteins that facilitates the understanding of unknown RNA functions and the creation of gene circuits and has potential for use in future therapeutics.

Crystal structure of endocrine-disrupting chemical bisphenol A and estrogen-related receptor γ.

The human estrogen-related receptor γ (ERRγ) is an orphan nuclear receptor. The ERRγ behaves as a constitutive activator of transcription and plays a key role in controlling mitochondrial energy production and energy metabolism. Bisphenol A (BPA) is used mainly in producing polycarbonate plastics and epoxy resins, but it is known as an endocrine disruptor and strongly binds to ERRγ. We determined the crystal structure of ERRγ in complex with BPA. Our structure revealed the molecular mechanism of BPA recognition by ERRγ, in which BPA is well anchored to its ligand-binding pocket. Our structure is the first report of the complex between a nuclear receptor and endocrine disruptor BPA. This structural analysis had a profound impact on subsequent studies of endocrine disruptors.

Non-conventional octameric structure of C-phycocyanin.

C-phycocyanin (CPC), a blue pigment protein, is an indispensable component of giant phycobilisomes, which are light-harvesting antenna complexes in cyanobacteria that transfer energy efficiently to photosystems I and II. X-ray crystallographic and electron microscopy (EM) analyses have revealed the structure of CPC to be a closed toroidal hexamer by assembling two trimers. In this study, the structural characterization of non-conventional octameric CPC is reported for the first time. Analyses of the crystal and cryogenic EM structures of the native CPC from filamentous thermophilic cyanobacterium Thermoleptolyngbya sp. O-77 unexpectedly illustrated the coexistence of conventional hexamer and novel octamer. In addition, an unusual dimeric state, observed via analytical ultracentrifugation, was postulated to be a key intermediate structure in the assemble of the previously unobserved octamer. These observations provide new insights into the assembly processes of CPCs and the mechanism of energy transfer in the light-harvesting complexes.

Minimal protein-only RNase P structure reveals insights into tRNA precursor recognition and catalysis.

Ribonuclease P (RNase P) is an endoribonuclease that catalyzes the processing of the 5' leader sequence of precursor tRNA (pre-tRNA). Ribonucleoprotein RNase P and protein-only RNase P (PRORP) in eukaryotes have been extensively studied, but the mechanism by which a prokaryotic nuclease recognizes and cleaves pre-tRNA is unclear. To gain insights into this mechanism, we studied homologs of Aquifex RNase P (HARPs), thought to be enzymes of approximately 23 kDa comprising only this nuclease domain. We determined the cryo-EM structure of Aq880, the first identified HARP enzyme. The structure unexpectedly revealed that Aq880 consists of both the nuclease and protruding helical (PrH) domains. Aq880 monomers assemble into a dimer via the PrH domain. Six dimers form a dodecamer with a left-handed one-turn superhelical structure. The structure also revealed that the active site of Aq880 is analogous to that of eukaryotic PRORPs. The pre-tRNA docking model demonstrated that 5' processing of pre-tRNAs is achieved by two adjacent dimers within the dodecamer. One dimer is responsible for catalysis, and the PrH domains of the other dimer are responsible for pre-tRNA elbow recognition. Our study suggests that HARPs measure an invariant distance from the pre-tRNA elbow to cleave the 5' leader sequence, which is analogous to the mechanism of eukaryotic PRORPs and the ribonucleoprotein RNase P. Collectively, these findings shed light on how different types of RNase P enzymes utilize the same pre-tRNA processing.

The crystal structure of mouse SULT2A8 reveals the mechanism of 7α-hydroxyl, bile acid sulfation.

Bile acids play essential roles in facilitating the intestinal absorption of lipophilic nutrients as well as regulation of glucose, lipid, and energy homeostasis via activation of some receptors. Bile acids are cytotoxic, and consequently their concentrations are tightly controlled. A critical pathway for bile acid elimination and detoxification is sulfation. The pattern of bile acid sulfation differs by species. Sulfation preferentially occurs at the 3α-OH of bile acids in humans, but at the 7α-OH in mice. A recent study identified mouse cytosolic sulfotransferase 2A8 (mSULT2A8) as the major hepatic 7α-hydroxyl bile acid-sulfating enzyme. To elucidate the 7α-OH specific sulfation mechanism of mSULT2A8, instead of 3α-OH specific sulfation in humans, we determined a crystal structure of mSULT2A8 in complex with cholic acid, a major bile acid, and 3'-phosphoadenosine-5'-phosphate, the sulfate donor product. Our study shows that bile acid-binding mode of mSULT2A8 and how the enzyme holds the 7α-OH group of bile acids at the catalytic center, revealing that the mechanism underlying 7α-OH specific sulfation. The structure shows the substrate binds to mSULT2A8 in an orientation perpendicular to that of human 3α-hydroxyl bile acid-sulfotransferase (hSULT2A1). The structure of the complex provides new insight into species different bile acid metabolism.

Pentatricopeptide repeats of protein-only RNase P use a distinct mode to recognize conserved bases and structural elements of pre-tRNA.

Pentatricopeptide repeat (PPR) motifs are α-helical structures known for their modular recognition of single-stranded RNA sequences with each motif in a tandem array binding to a single nucleotide. Protein-only RNase P 1 (PRORP1) in Arabidopsis thaliana is an endoribonuclease that uses its PPR domain to recognize precursor tRNAs (pre-tRNAs) as it catalyzes removal of the 5'-leader sequence from pre-tRNAs with its NYN metallonuclease domain. To gain insight into the mechanism by which PRORP1 recognizes tRNA, we determined a crystal structure of the PPR domain in complex with yeast tRNAPhe at 2.85 Å resolution. The PPR domain of PRORP1 bound to the structurally conserved elbow of tRNA and recognized conserved structural features of tRNAs using mechanisms that are different from the established single-stranded RNA recognition mode of PPR motifs. The PRORP1 PPR domain-tRNAPhe structure revealed a conformational change of the PPR domain upon tRNA binding and moreover demonstrated the need for pronounced overall flexibility in the PRORP1 enzyme conformation for substrate recognition and catalysis. The PRORP1 PPR motifs have evolved strategies for protein-tRNA interaction analogous to tRNA recognition by the RNA component of ribonucleoprotein RNase P and other catalytic RNAs, indicating convergence on a common solution for tRNA substrate recognition.

Nop9 recognizes structured and single-stranded RNA elements of preribosomal RNA.

Nop9 is an essential factor in the processing of preribosomal RNA. Its absence in yeast is lethal, and defects in the human ortholog are associated with breast cancer, autoimmunity, and learning/language impairment. PUF family RNA-binding proteins are best known for sequence-specific RNA recognition, and most contain eight α-helical repeats that bind to the RNA bases of single-stranded RNA. Nop9 is an unusual member of this family in that it contains eleven repeats and recognizes both RNA structure and sequence. Here we report a crystal structure of Saccharomyces cerevisiae Nop9 in complex with its target RNA within the 20S preribosomal RNA. This structure reveals that Nop9 brings together a carboxy-terminal module recognizing the 5' single-stranded region of the RNA and a bifunctional amino-terminal module recognizing the central double-stranded stem region. We further show that the 3' single-stranded region of the 20S target RNA adds sequence-independent binding energy to the RNA-Nop9 interaction. Both the amino- and carboxy-terminal modules retain the characteristic sequence-specific recognition of PUF proteins, but the amino-terminal module has also evolved a distinct interface, which allows Nop9 to recognize either single-stranded RNA sequences or RNAs with a combination of single-stranded and structured elements.

Biochemical and structural characterization of a thermostable Dps protein with His-type ferroxidase centers and outer metal-binding sites.

The DNA-binding protein from starved cells (Dps) is found in a wide range of microorganisms, and it has been well characterized. However, little is known about Dps proteins from nonheterocystous filamentous cyanobacteria. In this study, a Dps protein from the thermophilic nonheterocystous filamentous cyanobacterium Thermoleptolyngbya sp. O-77 (TlDps1) was purified and characterized. PAGE and CD analyses of TlDps1 demonstrated that it had higher thermostability than previously reported Dps proteins. X-ray crystallographic analysis revealed that TlDps1 possessed His-type ferroxidase centers within the cavity and unique metal-binding sites located on the surface of the protein, which presumably contributed to its exceedingly high thermostability.

Structural insight into the recognition of pathogen-derived phosphoglycolipids by C-type lectin receptor DCAR.

The C-type lectin receptors (CLRs) form a family of pattern recognition receptors that recognize numerous pathogens, such as bacteria and fungi, and trigger innate immune responses. The extracellular carbohydrate-recognition domain (CRD) of CLRs forms a globular structure that can coordinate a Ca2+ ion, allowing receptor interactions with sugar-containing ligands. Although well-conserved, the CRD fold can also display differences that directly affect the specificity of the receptors for their ligands. Here, we report crystal structures at 1.8-2.3 Å resolutions of the CRD of murine dendritic cell-immunoactivating receptor (DCAR, or Clec4b1), the CLR that binds phosphoglycolipids such as acylated phosphatidyl-myo-inositol mannosides (AcPIMs) of mycobacteria. Using mutagenesis analysis, we identified critical residues, Ala136 and Gln198, on the surface surrounding the ligand-binding site of DCAR, as well as an atypical Ca2+-binding motif (Glu-Pro-Ser/EPS168-170). By chemically synthesizing a water-soluble ligand analog, inositol-monophosphate dimannose (IPM2), we confirmed the direct interaction of DCAR with the polar moiety of AcPIMs by biolayer interferometry and co-crystallization approaches. We also observed a hydrophobic groove extending from the ligand-binding site that is in a suitable position to interact with the lipid portion of whole AcPIMs. These results suggest that the hydroxyl group-binding ability and hydrophobic groove of DCAR mediate its specific binding to pathogen-derived phosphoglycolipids such as mycobacterial AcPIMs.

Δ4-3-ketosteroids as a new class of substrates for the cytosolic sulfotransferases.

Cytosolic sulfotransferase (SULT)-mediated sulfation is generally known to involve the transfer of a sulfonate group from the active sulfate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), to a hydroxyl group or an amino group of a substrate compound. We report here that human SULT2A1, in addition to being able to sulfate dehydroepiandrosterone (DHEA) and other hydroxysteroids, could also catalyze the sulfation of Δ4-3-ketosteroids, which carry no hydroxyl groups in their chemical structure. Among a panel of Δ4-3-ketosteroids tested as substrates, 4-androstene-3,17-dione and progesterone were found to be sulfated by SULT2A1. Mass spectrometry analysis and structural modeling supported a reaction mechanism which involves the isomerization of Δ4-3-ketosteroids from the keto form to an enol form, prior to being subjected to sulfation. Results derived from this study suggested a potential role of SULT2A1 as a Δ4-3-ketosteroid sulfotransferase in steroid metabolism.

The Drosophila hnRNP F/H Homolog Glorund Uses Two Distinct RNA-Binding Modes to Diversify Target Recognition.

The Drosophila hnRNP F/H homolog, Glorund (Glo), regulates nanos mRNA translation by interacting with a structured UA-rich motif in the nanos 3' untranslated region. Glo regulates additional RNAs, however, and mammalian homologs bind G-tract sequences to regulate alternative splicing, suggesting that Glo also recognizes G-tract RNA. To gain insight into how Glo recognizes both structured UA-rich and G-tract RNAs, we used mutational analysis guided by crystal structures of Glo's RNA-binding domains and identified two discrete RNA-binding surfaces that allow Glo to recognize both RNA motifs. By engineering Glo variants that favor a single RNA-binding mode, we show that a subset of Glo's functions in vivo is mediated solely by the G-tract binding mode, whereas regulation of nanos requires both recognition modes. Our findings suggest a molecular mechanism for the evolution of dual RNA motif recognition in Glo that may be applied to understanding the functional diversity of other RNA-binding proteins.

The molecular basis for ANE syndrome revealed by the large ribosomal subunit processome interactome.

ANE syndrome is a ribosomopathy caused by a mutation in an RNA recognition motif of RBM28, a nucleolar protein conserved to yeast (Nop4). While patients with ANE syndrome have fewer mature ribosomes, it is unclear how this mutation disrupts ribosome assembly. Here we use yeast as a model system and show that the mutation confers growth and pre-rRNA processing defects. Recently, we found that Nop4 is a hub protein in the nucleolar large subunit (LSU) processome interactome. Here we demonstrate that the ANE syndrome mutation disrupts Nop4's hub function by abrogating several of Nop4's protein-protein interactions. Circular dichroism and NMR demonstrate that the ANE syndrome mutation in RRM3 of human RBM28 disrupts domain folding. We conclude that the ANE syndrome mutation generates defective protein folding which abrogates protein-protein interactions and causes faulty pre-LSU rRNA processing, thus revealing one aspect of the molecular basis of this human disease.

Crystal structure of human tyrosylprotein sulfotransferase-2 reveals the mechanism of protein tyrosine sulfation reaction.

Post-translational protein modification by tyrosine sulfation has an important role in extracellular protein-protein interactions. The protein tyrosine sulfation reaction is catalysed by the Golgi enzyme called the tyrosylprotein sulfotransferase. To date, no crystal structure is available for tyrosylprotein sulfotransferase. Detailed mechanism of protein tyrosine sulfation reaction has thus remained unclear. Here we present the first crystal structure of the human tyrosylprotein sulfotransferase isoform 2 complexed with a substrate peptide (C4P5Y3) derived from complement C4 and 3'-phosphoadenosine-5'-phosphate at 1.9 Å resolution. Structural and complementary mutational analyses revealed the molecular basis for catalysis being an SN2-like in-line displacement mechanism. Tyrosylprotein sulfotransferase isoform 2 appeared to recognize the C4 peptide in a deep cleft by using a short parallel ß-sheet type interaction, and the bound C4P5Y3 forms an L-shaped structure. Surprisingly, the mode of substrate peptide recognition observed in the tyrosylprotein sulfotransferase isoform 2 structure resembles that observed for the receptor type tyrosine kinases.