Investigating metabolic dysregulation in serum of triple transgenic Alzheimer's disease male mice: implications for pathogenesis and potential biomarkers.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disease that lacks convenient and accessible peripheral blood diagnostic markers and effective drugs. Metabolic dysfunction is one of AD risk factors, which leaded to alterations of various metabolites in the body. Pathological changes of the brain can be reflected in blood metabolites that are expected to explain the disease mechanisms or be candidate biomarkers. The aim of this study was to investigate the changes of targeted metabolites within peripheral blood of AD mouse model, with the purpose of exploring the disease mechanism and potential biomarkers. Targeted metabolomics was used to quantify 256 metabolites in serum of triple transgenic AD (3 × Tg-AD) male mice. Compared with controls, 49 differential metabolites represented dysregulation in purine, pyrimidine, tryptophan, cysteine and methionine and glycerophospholipid metabolism. Among them, adenosine, serotonin, N-acetyl-5-hydroxytryptamine, and acetylcholine play a key role in regulating neural transmitter network. The alteration of S-adenosine-L-homocysteine, S-adenosine-L-methionine, and trimethylamine-N-oxide in AD mice serum can served as indicator of AD risk. The results revealed the changes of metabolites in serum, suggesting that metabolic dysregulation in periphery in AD mice may be related to the disturbances in neuroinhibition, the serotonergic system, sleep function, the cholinergic system, and the gut microbiota. This study provides novel insights into the dysregulation of several key metabolites and metabolic pathways in AD, presenting potential avenues for future research and the development of peripheral biomarkers.

2'-O methylation of RNA cap in SARS-CoV-2 captured by serial crystallography.

The genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus has a capping modification at the 5'-untranslated region (UTR) to prevent its degradation by host nucleases. These modifications are performed by the Nsp10/14 and Nsp10/16 heterodimers using S-adenosylmethionine as the methyl donor. Nsp10/16 heterodimer is responsible for the methylation at the ribose 2'-O position of the first nucleotide. To investigate the conformational changes of the complex during 2'-O methyltransferase activity, we used a fixed-target serial synchrotron crystallography method at room temperature. We determined crystal structures of Nsp10/16 with substrates and products that revealed the states before and after methylation, occurring within the crystals during the experiments. Here we report the crystal structure of Nsp10/16 in complex with Cap-1 analog (m7GpppAm2'-O). Inhibition of Nsp16 activity may reduce viral proliferation, making this protein an attractive drug target.

NAD+ enhances the activity and thermostability of S-adenosyl-L-homocysteine hydrolase from Pyrococcus horikoshii OT3.

S-Adenosyl-L-methionine (SAM) and S-adenosyl-L-homocysteine (SAH) are important biochemical intermediates. SAM is the major methyl donor for diverse methylation reactions in vivo. The SAM to SAH ratio serves as a marker of methylation capacity. Stable isotope-labeled SAM and SAH are used to measure this ratio with high sensitivity. SAH hydrolase (EC 3.13.2.1; SAHH), which reversibly catalyzes the conversion of adenosine and L-homocysteine to SAH, is used to produce labeled SAH. To produce labeled SAH with high efficiency, we focused on the SAHH of Pyrococcus horikoshii OT3, a thermophilic archaeon. We prepared recombinant P. horikoshii SAHH using Escherichia coli and investigated its enzymatic properties. Unexpectedly, the optimum temperature and thermostability of P. horikoshii SAHH were much lower than its optimum growth temperature. However, addition of NAD+ to the reaction mixture shifted the optimum temperature of P. horikoshii SAHH to a higher temperature, suggesting that NAD+ stabilizes the structure of the enzyme.

Structural basis for substrate recognition by the human N-terminal methyltransferase 1.

α-N-terminal methylation represents a highly conserved and prevalent post-translational modification, yet its biological function has remained largely speculative. The recent discovery of α-N-terminal methyltransferase 1 (NTMT1) and its physiological substrates propels the elucidation of a general role of α-N-terminal methylation in mediating DNA-binding ability of the modified proteins. The phenotypes, observed from both NTMT1 knockdown in breast cancer cell lines and knockout mouse models, suggest the potential involvement of α-N-terminal methylation in DNA damage response and cancer development. In this study, we report the first crystal structures of human NTMT1 in complex with cofactor S-adenosyl-L-homocysteine (SAH) and six substrate peptides, respectively, and reveal that NTMT1 contains two characteristic structural elements (a ß hairpin and an N-terminal extension) that contribute to its substrate specificity. Our complex structures, coupled with mutagenesis, binding, and enzymatic studies, also present the key elements involved in locking the consensus substrate motif XPK (X indicates any residue type other than D/E) into the catalytic pocket for α-N-terminal methylation and explain why NTMT1 prefers an XPK sequence motif. We propose a catalytic mechanism for α-N-terminal methylation. Overall, this study gives us the first glimpse of the molecular mechanism of α-N-terminal methylation and potentially contributes to the advent of therapeutic agents for human diseases associated with deregulated α-N-terminal methylation.

Comparative Analysis of Sulfonium-π, Ammonium-π, and Sulfur-π Interactions and Relevance to SAM-Dependent Methyltransferases.

We report the measurement and analysis of sulfonium-π, thioether-π, and ammonium-π interactions in a ß-hairpin peptide model system, coupled with computational investigation and PDB analysis. These studies indicated that the sulfonium-π interaction is the strongest and that polarizability contributes to the stronger interaction with sulfonium relative to ammonium. Computational studies demonstrate that differences in solvation of the trimethylsulfonium versus the trimethylammonium group also contribute to the stronger sulfonium-π interaction. In comparing sulfonium-π versus sulfur-π interactions in proteins, analysis of SAM- and SAH-bound enzymes in the PDB suggests that aromatic residues are enriched in close proximity to the sulfur of both SAM and SAH, but the populations of aromatic interactions of the two cofactors are not significantly different, with the exception of the Me-π interactions in SAM, which are the most prevalent interaction in SAM but are not possible for SAH. This suggests that the weaker interaction energies due to loss of the cation-π interaction in going from SAM to SAH may contribute to turnover of the cofactor.

Crystal structure and ligand-induced folding of the SAM/SAH riboswitch.

While most SAM riboswitches strongly discriminate between SAM and SAH, the SAM/SAH riboswitch responds to both ligands with similar apparent affinities. We have determined crystal structures of the SAM/SAH riboswitch bound to SAH, SAM and other variant ligands at high resolution. The riboswitch forms an H-type pseudoknot structure with coaxial alignment of the stem-loop helix (P1) and the pseudoknot helix (PK). An additional three base pairs form at the non-open end of P1, and the ligand is bound at the interface between the P1 extension and the PK helix. The adenine nucleobase is stacked into the helix and forms a trans Hoogsteen-Watson-Crick base pair with a uridine, thus becoming an integral part of the helical structure. The majority of the specific interactions are formed with the adenosine. The methionine or homocysteine chain lies in the groove making a single hydrogen bond, and there is no discrimination between the sulfonium of SAM or the thioether of SAH. Single-molecule FRET analysis reveals that the riboswitch exists in two distinct conformations, and that addition of SAM or SAH shifts the population into a stable state that likely corresponds to the form observed in the crystal. A model for translational regulation is presented whereby in the absence of ligand the riboswitch is largely unfolded, lacking the PK helix so that translation can be initiated at the ribosome binding site. But the presence of ligand stabilizes the folded conformation that includes the PK helix, so occluding the ribosome binding site and thus preventing the initiation of translation.

Crystal structure of human cytoplasmic tRNAHis-specific 5'-monomethylphosphate capping enzyme.

BCDIN3 domain containing RNA methyltransferase, BCDIN3D, monomethylates the 5'-monophosphate of cytoplasmic tRNAHis with a G-1:A73 mispair at the top of an eight-nucleotide-long acceptor helix, using S-adenosyl-l-methionine (SAM) as a methyl group donor. In humans, BCDIN3D overexpression is associated with the tumorigenic phenotype and poor prognosis in breast cancer. Here, we present the crystal structure of human BCDIN3D complexed with S-adenosyl-l-homocysteine. BCDIN3D adopts a classical Rossmann-fold methyltransferase structure. A comparison of the structure with that of the closely related methylphosphate capping enzyme, MePCE, which monomethylates the 5'-γ-phosphate of 7SK RNA, revealed the important residues for monomethyl transfer from SAM onto the 5'-monophosphate of tRNAHis and for tRNAHis recognition by BCDIN3D. A structural model of tRNAHis docking onto BCDIN3D suggested the molecular mechanism underlying the different activities between BCDIN3D and MePCE. A loop in BCDIN3D is shorter, as compared to the corresponding region that forms an α-helix to recognize the 5'-end of RNA in MePCE, and the G-1:A73 mispair in tRNAHis allows the N-terminal α-helix of BCDIN3D to wedge the G-1:A73 mispair of tRNAHis. As a result, the 5'-monophosphate of G-1 of tRNAHis is deep in the catalytic pocket for 5'-phosphate methylation. Thus, BCDIN3D is a tRNAHis-specific 5'-monomethylphosphate capping enzyme that discriminates tRNAHis from other tRNA species, and the structural information presented in this study also provides the molecular basis for the development of drugs against breast cancers.

Crystal structure of a S-adenosyl-L-methionine-dependent O-methyltransferase-like enzyme from Aspergillus flavus.

S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTases) are widely distributed among almost all organisms and often characterized with conserved Rossmann fold, TIM barrel, and D×G×G×G motif. However, some MTases show no methyltransferase activity. In the present study, the crystal structure of LepI, one MTase-like enzyme isolated from A. flavus that catalyzes pericyclic reactions, was investigated to determine its structure-function relationship. The overall structure of LepI in complex with the SAM mimic S-adenosyl-L-homocysteine (SAH) (PDB ID: 6IV7) indicated that LepI is a tetramer in solution. The residues His133, Arg197, Arg295, and Asp296 located near the active site can form hydrogen bonds with the substrate, thus participating in catalytic reactions. The binding of SAH in LepI is almost identical to that in other resolved MTases; however, the location of catalytic residues differs significantly. Phylogenetic trials suggest that LepI proteins share a common ancestor in plants and algae, which may explain the conserved SAM-binding site. However, the accelerated evolution of A. flavus has introduced both functional and structural changes in LepI. More importantly, the residue Arg295, which is unique to LepI, might be a key determinant for the altered enzymatic behavior. Collectively, the differences in the composition of catalytic residues, as well as the unique tetrameric form of LepI, define its unique enzymatic behavior. The present work provides an additional understanding of the structure-function relationship of MTases and MTase-like enzymes.

The structure of the SAM/SAH-binding riboswitch.

S-adenosylmethionine (SAM) is a central metabolite since it is used as a methyl group donor in many different biochemical reactions. Many bacteria control intracellular SAM concentrations using riboswitch-based mechanisms. A number of structurally different riboswitch families specifically bind to SAM and mainly regulate the transcription or the translation of SAM-biosynthetic enzymes. In addition, a highly specific riboswitch class recognizes S-adenosylhomocysteine (SAH)-the product of SAM-dependent methyl group transfer reactions-and regulates enzymes responsible for SAH hydrolysis. High-resolution structures are available for many of these riboswitch classes and illustrate how they discriminate between the two structurally similar ligands SAM and SAH. The so-called SAM/SAH riboswitch class binds both ligands with similar affinities and is structurally not yet characterized. Here, we present a high-resolution nuclear magnetic resonance structure of a member of the SAM/SAH-riboswitch class in complex with SAH. Ligand binding induces pseudoknot formation and sequestration of the ribosome binding site. Thus, the SAM/SAH-riboswitches are translational 'OFF'-switches. Our results establish a structural basis for the unusual bispecificity of this riboswitch class. In conjunction with genomic data our structure suggests that the SAM/SAH-riboswitches might be an evolutionary late invention and not a remnant of a primordial RNA-world as suggested for other riboswitches.

Mechanism of allosteric activation of human mRNA cap methyltransferase (RNMT) by RAM: insights from accelerated molecular dynamics simulations.

The RNA guanine-N7 methyltransferase (RNMT) in complex with RNMT-activating miniprotein (RAM) catalyses the formation of a N7-methylated guanosine cap structure on the 5' end of nascent RNA polymerase II transcripts. The mRNA cap protects the primary transcript from exonucleases and recruits cap-binding complexes that mediate RNA processing, export and translation. By using microsecond standard and accelerated molecular dynamics simulations, we provide for the first time a detailed molecular mechanism of allosteric regulation of RNMT by RAM. We show that RAM selects the RNMT active site conformations that are optimal for binding of substrates (AdoMet and the cap), thus enhancing their affinity. Furthermore, our results strongly suggest the likely scenario in which the cap binding promotes the subsequent AdoMet binding, consistent with the previously suggested cooperative binding model. By employing the network community analyses, we revealed the underlying long-range allosteric networks and paths that are crucial for allosteric regulation by RAM. Our findings complement and explain previous experimental data on RNMT activity. Moreover, this study provides the most complete description of the cap and AdoMet binding poses and interactions within the enzyme's active site. This information is critical for the drug discovery efforts that consider RNMT as a promising anti-cancer target.

Structural Basis of the Substrate Selectivity of Viperin.

Viperin is a radical S-adenosylmethionine (SAM) enzyme that inhibits viral replication by converting cytidine triphosphate (CTP) into 3'-deoxy-3',4'-didehydro-CTP and by additional undefined mechanisms operating through its N- and C-terminal domains. Here, we describe crystal structures of viperin bound to a SAM analogue and CTP or uridine triphosphate (UTP) and report kinetic parameters for viperin-catalyzed reactions with CTP or UTP as substrates. Viperin orients the C4' hydrogen atom of CTP and UTP similarly for abstraction by a 5'-deoxyadenosyl radical, but the uracil moiety introduces unfavorable interactions that prevent tight binding of UTP. Consistently, kcat is similar for CTP and UTP whereas the Km for UTP is much greater. The structures also show that nucleotide binding results in ordering of the C-terminal tail and reveal that this region contains a P-loop that binds the γ-phosphate of the bound nucleotide. Collectively, the results explain the selectivity for CTP and reveal a structural role for the C-terminal tail in binding CTP and UTP.

Novel parameter describing restriction endonucleases: Secondary-Cognate-Specificity and chemical stimulation of TsoI leading to substrate specificity change.

Over 470 prototype Type II restriction endonucleases (REases) are currently known. Most recognise specific DNA sequences 4-8 bp long, with very few exceptions cleaving DNA more frequently. TsoI is a thermostable Type IIC enzyme that recognises the DNA sequence TARCCA (R = A or G) and cleaves downstream at N11/N9. The enzyme exhibits extensive top-strand nicking of the supercoiled single-site DNA substrate. The second DNA strand of such substrate is specifically cleaved only in the presence of duplex oligonucleotides containing a cognate site. We have previously shown that some Type IIC/IIG/IIS enzymes from the Thermus-family exhibit 'affinity star' activity, which can be induced by the S-adenosyl-L-methionine (SAM) cofactor analogue-sinefungin (SIN). Here, we define a novel type of inherently built-in 'star' activity, exemplified by TsoI. The TsoI 'star' activity cannot be described under the definition of the classic 'star' activity as it is independent of the reaction conditions used and cannot be separated from the cognate specificity. Therefore, we define this phenomenon as Secondary-Cognate-Specificity (SCS). The TsoI SCS comprises several degenerated variants of the cognate site. Although the efficiency of TsoI SCS cleavage is lower in comparison to the cognate TsoI recognition sequence, it can be stimulated by S-adenosyl-L-cysteine (SAC). We present a new route for the chemical synthesis of SAC. The TsoI/SAC REase may serve as a novel tool for DNA manipulation.

Mechanistic features of the atypical tRNA m1G9 SPOUT methyltransferase, Trm10.

The tRNA m1G9 methyltransferase (Trm10) is a member of the SpoU-TrmD (SPOUT) superfamily of methyltransferases, and Trm10 homologs are widely conserved throughout Eukarya and Archaea. Despite possessing the trefoil knot characteristic of SPOUT enzymes, Trm10 does not share the same quaternary structure or key sequences with other members of the SPOUT family, suggesting a novel mechanism of catalysis. To investigate the mechanism of m1G9 methylation by Trm10, we performed a biochemical and kinetic analysis of Trm10 and variants with alterations in highly conserved residues, using crystal structures solved in the absence of tRNA as a guide. Here we demonstrate that a previously proposed general base residue (D210 in Saccharomyces cerevisiae Trm10) is not likely to play this suggested role in the chemistry of methylation. Instead, pH-rate analysis suggests that D210 and other conserved carboxylate-containing residues at the active site collaborate to establish an active site environment that promotes a single ionization that is required for catalysis. Moreover, Trm10 does not depend on a catalytic metal ion, further distinguishing it from the other known SPOUT m1G methyltransferase, TrmD. These results provide evidence for a non-canonical tRNA methyltransferase mechanism that characterizes the Trm10 enzyme family.

Neutron structures of the Helicobacter pylori 5'-methylthioadenosine nucleosidase highlight proton sharing and protonation states.

MTAN (5'-methylthioadenosine nucleosidase) catalyzes the hydrolysis of the N-ribosidic bond of a variety of adenosine-containing metabolites. The Helicobacter pylori MTAN (HpMTAN) hydrolyzes 6-amino-6-deoxyfutalosine in the second step of the alternative menaquinone biosynthetic pathway. Substrate binding of the adenine moiety is mediated almost exclusively by hydrogen bonds, and the proposed catalytic mechanism requires multiple proton-transfer events. Of particular interest is the protonation state of residue D198, which possesses a pKa above 8 and functions as a general acid to initiate the enzymatic reaction. In this study we present three corefined neutron/X-ray crystal structures of wild-type HpMTAN cocrystallized with S-adenosylhomocysteine (SAH), Formycin A (FMA), and (3R,4S)-4-(4-Chlorophenylthiomethyl)-1-[(9-deaza-adenin-9-yl)methyl]-3-hydroxypyrrolidine (p-ClPh-Thio-DADMe-ImmA) as well as one neutron/X-ray crystal structure of an inactive variant (HpMTAN-D198N) cocrystallized with SAH. These results support a mechanism of D198 pKa elevation through the unexpected sharing of a proton with atom N7 of the adenine moiety possessing unconventional hydrogen-bond geometry. Additionally, the neutron structures also highlight active site features that promote the stabilization of the transition state and slight variations in these interactions that result in 100-fold difference in binding affinities between the DADMe-ImmA and ImmA analogs.

A glutamate/aspartate switch controls product specificity in a protein arginine methyltransferase.

Trypanosoma brucei PRMT7 (TbPRMT7) is a protein arginine methyltransferase (PRMT) that strictly monomethylates various substrates, thus classifying it as a type III PRMT. However, the molecular basis of its unique product specificity has remained elusive. Here, we present the structure of TbPRMT7 in complex with its cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.8 Å resolution and identify a glutamate residue critical for its monomethylation behavior. TbPRMT7 comprises the conserved methyltransferase and ß-barrel domains, an N-terminal extension, and a dimerization arm. The active site at the interface of the N-terminal extension, methyltransferase, and ß-barrel domains is stabilized by the dimerization arm of the neighboring protomer, providing a structural basis for dimerization as a prerequisite for catalytic activity. Mutagenesis of active-site residues highlights the importance of Glu181, the second of the two invariant glutamate residues of the double E loop that coordinate the target arginine in substrate peptides/proteins and that increase its nucleophilicity. Strikingly, mutation of Glu181 to aspartate converts TbPRMT7 into a type I PRMT, producing asymmetric dimethylarginine (ADMA). Isothermal titration calorimetry (ITC) using a histone H4 peptide showed that the Glu181Asp mutant has markedly increased affinity for monomethylated peptide with respect to the WT, suggesting that the enlarged active site can favorably accommodate monomethylated peptide and provide sufficient space for ADMA formation. In conclusion, these findings yield valuable insights into the product specificity and the catalytic mechanism of protein arginine methyltransferases and have important implications for the rational (re)design of PRMTs.

The Molecular and Structural Basis of O-methylation Reaction in Coumarin Biosynthesis in Peucedanum praeruptorum Dunn.

Methoxylated coumarins represent a large proportion of officinal value coumarins while only one enzyme specific to bergaptol O-methylation (BMT) has been identified to date. The multiple types of methoxylated coumarins indicate that at least one unknown enzyme participates in the O-methylation of other hydroxylated coumarins and remains to be identified. Combined transcriptome and metabonomics analysis revealed that an enzyme similar to caffeic acid O-methyltransferase (COMT-S, S is short for similar) was involved in catalyzing all the hydroxylated coumarins in Peucedanum praeruptorum. However, the precise molecular mechanism of its substrate heterozygosis remains unsolved. Pursuing this question, we determined the crystal structure of COMT-S to clarify its substrate preference. The result revealed that Asn132, Asp271, and Asn325 govern the substrate heterozygosis of COMT-S. A single mutation, such as N132A, determines the catalytic selectivity of hydroxyl groups in esculetin and also causes production differences in bergapten. Evolution-based analysis indicated that BMT was only recently derived as a paralogue of caffeic acid O-methyltransferase (COMT) via gene duplication, occurring before the Apiaceae family divergence between 37 and 100 mya. The present study identified the previously unknown O-methylation steps in coumarin biosynthesis. The crystallographic and mutational studies provided a deeper understanding of the substrate preference, which can be used for producing specific O-methylation coumarins. Moreover, the evolutionary relationship between BMT and COMT-S was clarified to facilitate understanding of evolutionary events in the Apiaceae family.

Water-Mediated Carbon-Oxygen Hydrogen Bonding Facilitates S-Adenosylmethionine Recognition in the Reactivation Domain of Cobalamin-Dependent Methionine Synthase.

The C-terminal domain of cobalamin-dependent methionine synthase (MetH) has an essential role in catalyzing the reactivation of the enzyme following the oxidation of its cobalamin cofactor. This reactivation occurs through reductive methylation of the cobalamin using S-adenosylmethionine (AdoMet) as the methyl donor. Herein, we examine the molecular recognition of AdoMet by the MetH reactivation domain utilizing structural, biochemical, and computational approaches. Crystal structures of the Escherichia coli MetH reactivation domain in complex with AdoMet, the methyl transfer product S-adenosylhomocysteine (AdoHcy), and the AdoMet analogue inhibitor sinefungin illustrate that the ligands exhibit an analogous conformation within the solvent-exposed substrate binding cleft of the enzyme. AdoMet binding is stabilized by an intramolecular sulfur-oxygen chalcogen bond between the sulfonium and carboxylate groups of the substrate and by water-mediated carbon-oxygen hydrogen bonding between the sulfonium cation and the side chains of Glu1097 and Glu1128 that bracket the substrate binding cleft. AdoMet and sinefungin exhibited similar binding affinities for the MetH reactivation domain, whereas AdoHcy displayed an affinity for the enzyme that was an order of magnitude lower. Mutations of Glu1097 and Glu1128 diminished the AdoMet/AdoHcy binding selectivity ratio to approximately 2-fold, underscoring the role of these residues in enabling the enzyme to discriminate between the substrate and product. Together, these findings indicate that Glu1097 and Glu1128 in MetH promote high-affinity recognition of AdoMet and that sinefungin and potentially other AdoMet-based methyltransferase inhibitors can abrogate MetH reactivation, which would result in off-target effects associated with alterations in methionine homeostasis and one-carbon metabolism.

Recent Advances in Chemical Tools for the Regulation and Study of Protein Lysine Methyltransferases.

Protein lysine methyltransferases (PKMTs) are epigenetic regulators that modulate gene transcription and physiological functions by catalyzing the post-translational methylation of specific lysine residues of substrate proteins, such as histones. They are considered to be candidate drugs for the treatment of various diseases, including acute myeloid leukemia, and in the past decade, potent and selective inhibitors of individual PKMTs have been developed. Some are currently under clinical trial. In this review, we will focus on some breakthrough PKMT inhibitors, and discuss chemistry-based methods available for elucidation of the physiological functions of PKMTs and methylated proteins.

Biochemical Characterization and Structural Basis of Reactivity and Regioselectivity Differences between Burkholderia thailandensis and Burkholderia glumae 1,6-Didesmethyltoxoflavin N-Methyltransferase.

Burkholderia glumae converts the guanine base of guanosine triphosphate into an azapteridine and methylates both the pyrimidine and triazine rings to make toxoflavin. Strains of Burkholderia thailandensis and Burkholderia pseudomallei have a gene cluster encoding seven putative biosynthetic enzymes that resembles the toxoflavin gene cluster. Four of the enzymes are similar in sequence to BgToxBCDE, which have been proposed to make 1,6-didesmethyltoxoflavin (1,6-DDMT). One of the remaining enzymes, BthII1283 in B. thailandensis E264, is a predicted S-adenosylmethionine (SAM)-dependent N-methyltransferase that shows a low level of sequence identity to BgToxA, which sequentially methylates N6 and N1 of 1,6-DDMT to form toxoflavin. Here we show that, unlike BgToxA, BthII1283 catalyzes a single methyl transfer to N1 of 1,6-DDMT in vitro. In addition, we investigated the differences in reactivity and regioselectivity by determining crystal structures of BthII1283 with bound S-adenosylhomocysteine (SAH) or 1,6-DDMT and SAH. BthII1283 contains a class I methyltransferase fold and three unique extensions used for 1,6-DDMT recognition. The active site structure suggests that 1,6-DDMT is bound in a reduced form. The plane of the azapteridine ring system is orthogonal to its orientation in BgToxA. In BthII1283, the modeled SAM methyl group is directed toward the p orbital of N1, whereas in BgToxA, it is first directed toward an sp2 orbital of N6 and then toward an sp2 orbital of N1 after planar rotation of the azapteridine ring system. Furthermore, in BthII1283, N1 is hydrogen bonded to a histidine residue whereas BgToxA does not supply an obvious basic residue for either N6 or N1 methylation.

Transient Kinetics Define a Complete Kinetic Model for Protein Arginine Methyltransferase 1.

Protein arginine methyltransferases (PRMTs) are the enzymes responsible for posttranslational methylation of protein arginine residues in eukaryotic cells, particularly within the histone tails. A detailed mechanistic model of PRMT-catalyzed methylation is currently lacking, but it is essential for understanding the functions of PRMTs in various cellular pathways and for efficient design of PRMT inhibitors as potential treatments for a range of human diseases. In this work, we used stopped-flow fluorescence in combination with global kinetic simulation to dissect the transient kinetics of PRMT1, the predominant type I arginine methyltransferase. Several important mechanistic insights were revealed. The cofactor and the peptide substrate bound to PRMT1 in a random manner and then followed a kinetically preferred pathway to generate the catalytic enzyme-cofactor-substrate ternary complex. Product release proceeded in an ordered fashion, with peptide dissociation followed by release of the byproduct S-adenosylhomocysteine. Importantly, the dissociation rate of the monomethylated intermediate from the ternary complex was much faster than the methyl transfer. Such a result provided direct evidence for distributive arginine dimethylation, which means the monomethylated substrate has to be released to solution and rebind with PRMT1 before it undergoes further methylation. In addition, cofactor binding involved a conformational transition, likely an open-to-closed conversion of the active site pocket. Further, the histone H4 peptide bound to the two active sites of the PRMT1 homodimer with differential affinities, suggesting a negative cooperativity mechanism of substrate binding. These findings provide a new mechanistic understanding of how PRMTs interact with their substrates and transfer methyl groups.