Methyltransferase Domain Research Articles

Expression and purification of the truncated duck DTMUV NS5 protein and the subcellular localization of NS5 in vitro

Duck Tembusu virus (DTMUV) non-structural protein 5 (NS5), which harbors an N-terminal methyltransferase (MTase) domain and a C-terminal RNA-dependent RNA polymerase (RdRp) domain, is central to virus replication. In this study, a sequence encoding amino acid residues 1-408 of NS5 was amplified and ligated into a pET32a vector to generate a pET32a-NS51-408 expression vector. Recombinant protein was expressed, purified, and used to arise a mouse anti-NS5 specific polyclonal antibody (DTMUV-NS51-408 mPAb). Indirect immunofluorescence assays indicated that NS5 protein localized within the cytoplasmic region of DTMUV-infected or transfected cells; the localization was not affected in the presence of a nuclear export inhibitor. This study provides the first demonstration that, in contrast to the data reported for NS5 proteins of Dengue and Yellow fever virus, DTMUV NS5 had slight nuclear localization activity.

Chimeric 6-methylsalicylic acid synthase with domains of acyl carrier protein and methyltransferase from Pseudallescheria boydii shows novel biosynthetic activity.

SummaryPolyketides are important secondary metabolites, many of which exhibit potent pharmacological applications. Biosynthesis of polyketides is carried out by a single polyketide synthase (PKS) or multiple PKSs in successive elongations of enzyme‐bound intermediates related to fatty acid biosynthesis. The polyketide gene PKS306 from Pseudallescheria boydii NTOU2362 containing domains of ketosynthase (KS), acyltransferase (AT), dehydratase (DH), acyl carrier protein (ACP) and methyltransferase (MT) was cloned in an attempt to produce novel chemical compounds, and this PKS harbouring green fluorescent protein (GFP) was expressed in Saccharomyces cerevisiae. Although fluorescence of GFP and fusion protein analysed by anti‐GFP antibody were observed, no novel compound was detected. 6‐methylsalicylic acid synthase (6MSAS) was then used as a template and engineered with PKS306 by combinatorial fusion. The chimeric PKS containing domains of KS, AT, DH and ketoreductase (KR) from 6MSAS with ACP and MT from PKS306 demonstrated biosynthesis of a novel compound. The compound was identified with a deduced chemical formula of C7H10O3, and the chemical structure was named as 2‐hydroxy‐2‐(propan‐2‐yl) cyclobutane‐1,3‐dione. The novel compound synthesized by the chimeric PKS in this study demonstrates the feasibility of combinatorial fusion of PKS genes to produce novel polyketides.

Structure of the yellow fever NS5 protein reveals conserved drug targets shared among flaviviruses

Yellow fever virus (YFV) is responsible for devastating outbreaks of Yellow fever (YF) in humans and is associated with high mortality rates. Recent large epidemics and epizootics and exponential increases in the numbers of YF cases in humans and non-human primates highlight the increasing threat YFV poses, despite the availability of an effective YFV vaccine. YFV is the first human virus discovered, but the structures of several of the viral proteins remain poorly understood. Here we report the structure of the full-length NS5 protein, a key enzyme for the replication of flaviviruses that contains both a methyltransferase domain and an RNA dependent RNA polymerase domain, at 3.1 Å resolution. The viral polymerase adopts right-hand fold, demonstrating the similarities of the Yellow fever, Dengue and Zika polymerases. Together this data suggests NS5 as a prime and ideal target for the design of pan-flavivirus inhibitors.

Biochemical Characterization of the Methylmercaptopropionate:Cob(I)alamin Methyltransferase from Methanosarcina acetivorans.

Methanogenesis from methylated substrates is initiated by substrate-specific methyltransferases that generate the central metabolic intermediate methyl-coenzyme M. This reaction involves a methyl-corrinoid protein intermediate and one or two cognate methyltransferases. Based on genetic data, the Methanosarcina acetivorans MtpC (corrinoid protein) and MtpA (methyltransferase) proteins were suggested to catalyze the methylmercaptopropionate (MMPA):coenzyme M (CoM) methyl transfer reaction without a second methyltransferase. To test this, MtpA was purified after overexpression in its native host and characterized biochemically. MtpA catalyzes a robust methyl transfer reaction using free methylcob(III)alamin as the donor and mercaptopropionate (MPA) as the acceptor, with kcat of 0.315 s-1 and apparent Km for MPA of 12 μM. CoM did not serve as a methyl acceptor; thus, a second unidentified methyltransferase is required to catalyze the full MMPA:CoM methyl transfer reaction. The physiologically relevant methylation of cob(I)alamin with MMPA, which is thermodynamically unfavorable, was also demonstrated, but only at high substrate concentrations. Methylation of cob(I)alamin with methanol, dimethylsulfide, dimethylamine, and methyl-CoM was not observed, even at high substrate concentrations. Although the corrinoid protein MtpC was poorly expressed alone, a stable MtpA/MtpC complex was obtained when both proteins were coexpressed. Biochemical characterization of this complex was not feasible, because the corrinoid cofactor of this complex was in the inactive Co(II) state and was not reactivated by incubation with strong reductants. The MtsF protein, composed of both corrinoid and methyltransferase domains, copurifies with the MtpA/MtpC, suggesting that it may be involved in MMPA metabolism.IMPORTANCE Methylmercaptopropionate (MMPA) is an environmentally significant molecule produced by degradation of the abundant marine metabolite dimethylsulfoniopropionate, which plays a significant role in the biogeochemical cycles of both carbon and sulfur, with ramifications for ecosystem productivity and climate homeostasis. Detailed knowledge of the mechanisms for MMPA production and consumption is key to understanding steady-state levels of this compound in the biosphere. Unfortunately, the biochemistry required for MMPA catabolism under anoxic conditions is poorly characterized. The data reported here validate the suggestion that the MtpA protein catalyzes the first step in the methanogenic catabolism of MMPA. However, the enzyme does not catalyze a proposed second step required to produce the key intermediate, methyl coenzyme M. Therefore, the additional enzymes required for methanogenic MMPA catabolism await discovery.

Suppression of human STAT2 by ZIKV NS5

Abstract The infection of ZIKA virus (ZIKV), a mosquito-transmitted flavivirus, has been shown to cause congenital disease in the context of infection during pregnancy in its 2015 outbreak. To establish the infection, ZIKV must suppress host interferon (IFN) response. Previous studies have shown that ZIKV utilizes its non-structural NS5 protein to trigger the human STAT2, a critical mediator to boost host interferon (IFN) response, for the proteasome-dependent degradation. However, the underlying mechanisms of viral NS5 and hSTAT2 remain elusive largely due to the lack of atom level conformational information. Here, we report the structure of hSTAT2 in complex with ZIKV NS5. hSTAT2, with the SH2 domain occluded by the C-terminal tyrosine-phosphorylation (pY)-tail segment, inserts the coiled-coil domain into the cleft between the methyltransferase and RdRP domains of NS5 in a manner that would occlude the association of hSTAT2 with cellular regulatory protein IRF9. Disruption of the ZIKV NS5 – hSTAT2 association in cells compromises the NS5-mediated hSTAT2 degradation and IFN responses. This study provides a basis for understanding the functional antagonism between viral antagonists and STAT2.

Supramolecular arrangement of the full-length Zika virus NS5.

Zika virus (ZIKV), a member of the Flaviviridae family, has emerged as a major public health threat, since ZIKV infection has been connected to microcephaly and other neurological disorders. Flavivirus genome replication is driven by NS5, an RNA-dependent RNA polymerase (RdRP) that also contains a N-terminal methyltransferase domain essential for viral mRNA capping. Given its crucial roles, ZIKV NS5 has become an attractive antiviral target. Here, we have used integrated structural biology approaches to characterize the supramolecular arrangement of the full-length ZIKV NS5, highlighting the assembly and interfaces between NS5 monomers within a dimeric structure, as well as the dimer-dimer interactions to form higher order fibril-like structures. The relative orientation of each monomer within the dimer provides a model to explain the coordination between MTase and RdRP domains across neighboring NS5 molecules and mutational studies underscore the crucial role of the MTase residues Y25, K28 and K29 in NS5 dimerization. The basic residue K28 also participates in GTP binding and competition experiments indicate that NS5 dimerization is disrupted at high GTP concentrations. This competition represents a first glimpse at a molecular level explaining how dimerization might regulate the capping process.

Investigation into the Role of the Set2 Auto‐Inhibitory Domain in H3K36 Methyltransferase Activity

Set2 is nucleosomal‐specific methyltransferase that is responsible for mediating histone H3 lysine 36 (H3K36) mono‐, di‐, or trimethylation in Saccharomyces cerevisiae. The human homolog of Set2, SETD2, mediates only H3K36me3. H3K36 methylation has been linked to the prevention of inappropriate or “cryptic” transcription, and SETD2 functions as a tumor repressor through its regulation of the cell cycle and mitotic function. While Set2 catalyzes H3K36 methylation via its SET methyltransferase domain located at its N‐terminus, Set2 contains a Set2‐Rpb1 interaction (SRI) domain at its C‐terminus that binds RNA polymerase II and couples H3K36 methylation with transcriptional elongation. In addition to the SET and SRI domains, a third domain termed the auto inhibitory domain (AID) was identified within Set2 and functions to control the extent of methylation and substrate specificity of Set2's H3K36 methyltransferase activity (1). Additionally, a new study revealed key residues within the AID that are required for the autoinhibitory function of this domain (2). Here, we used an in vitro biochemical approach to further examine the role of the AID in Set2 methyltransferase activity. We designed and expressed full‐length (FL) Set2 containing a recently characterized mutation of the AID (i.e., H336Y) (2) as well as the AID in isolation to test the possibility that this domain directly interacts with the SET domain of Set2 to regulate its H3K36 methylation. Consistent with previous reports, we found that the H336Y AID mutation in Set2 resulted in significantly higher H3K36 methylation activity on nucleosomal and core histone substrates in vitro. Importantly, the normal preference of Set2 for nucleosomes over core histones was lost in the H366Y AID mutation; i.e., the H366Y AID mutation in Set2 enhanced core histone methylation activity to those found with oligonucleosomes. Examination of the AID's ability to interact with Set2's SET domain to regulate its H3K36 methyltransferase activity was next examined. Contrary to our predictions, we found that the addition of AID in Set2 methyltransferase assays did not significantly alter Set2's methyltransferase activity on either oligonucleosomes or core histones. Further preliminary co‐immunoprecipitation analyses did not support a direct interaction of the AID with Set2. These data suggest that the AID's repressive action on Set2 may be regulated through a unique AID structure that exists adjacently to the SET domain which enforces substrate preference and activity rather than by a scenario where the AID interacts directly with, and occludes, the SET domain. Taken together, our data extends our understanding of the autoinhibition functions found in chromatin regulators that direct the precise transcriptional control between chromatin accessibility and repression.Support or Funding InformationNIH to BDSThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metagenomes of a Freshwater Charavirus from British Columbia Provide a Window into Ancient Lineages of Viruses.

Charophyte algae, not chlorophyte algae, are the ancestors of ‘higher plants’; hence, viruses infecting charophytes may be related to those that first infected higher plants. Streamwaters from British Columbia, Canada, yielded single-stranded RNA metagenomes of Charavirus canadensis (CV-Can), that are similar in genomic architecture, length (9593 nt), nucleotide identity (63.4%), and encoded amino-acid sequence identity (53.0%) to those of Charavirus australis (CV-Aus). The sequences of their RNA-dependent RNA-polymerases (RdRp) resemble those found in benyviruses, their helicases those of hepaciviruses and hepegiviruses, and their coat-proteins (CP) those of tobamoviruses; all from the alphavirus/flavivirus branch of the ‘global RNA virome’. The 5’-terminus of the CV-Can genome, but not that of CV-Aus, is complete and encodes a methyltransferase domain. Comparisons of CP sequences suggests that Canadian and Australian charaviruses diverged 29–46 million years ago (mya); whereas, the CPs of charaviruses and tobamoviruses last shared a common ancestor 212 mya, and the RdRps of charaviruses and benyviruses 396 mya. CV-Can is sporadically abundant in low-nutrient freshwater rivers in British Columbia, where Chara braunii, a close relative of C. australis, occurs, and which may be its natural host. Charaviruses, like their hosts, are ancient and widely distributed, and thus provide a window to the viromes of early eukaryotes and, even, Archaea.

The Crg1 N-Terminus Is Essential for Methyltransferase Activity and Cantharidin Resistance in Saccharomyces cerevisiae.

Crg1 is an S-adenosylmethionine (SAM)-dependent methyltransferase required for cantharidin resistance in yeast. Crg1 has a well-characterized methyltransferase domain that inactivates cantharidin by methylation. However, the remaining part of the Crg1 protein is yet to be functionally characterized. In this study, we identified an essential role of the N-terminus of Crg1 in methyltransferase activity and cantharidin resistance. Yeast cells lacking 41 residues of the N-terminus of Crg1 ( crg1ΔN) showed hypersensitivity to cantharidin as same as the null mutant, crg1. The mass spectrometry-based biochemical enzyme assay revealed a loss of methyltransferase activity in Crg1ΔN, which justifies the loss of cantharidin resistance, as well. The subcellular distribution of Crg1ΔN-daGFP showed cytoplasmic aggregates, whereas wild-type Crg1-daGFP was distributed normally in the cytoplasm. Interestingly, the Crg1-methyltransferase domain point mutants (D44A, D67A, and E105A/D108A) also showed the same cytoplasmic aggregates as Crg1ΔN-daGFP. In silico prediction of the tertiary structures of these mutants indicated an altered protein conformation. Altogether, these observations suggest that the N-terminal truncation, as well as the point mutations in the methyltransferase domain, alters the native folding of Crg1 methyltransferase, resulting in a loss of enzyme activity. Furthermore, the crg1ΔN mutant showed the same phenotypes as the crg1 null mutant in the presence of cantharidin, i.e., lethal cell growth, PE auxotrophy, temperature sensitivity, endoplasmic reticulum stress, GPI anchor missorting, and cell wall damage. Overall, this study identifies an essential role of the N-terminus of Crg1 in methyltransferase activity and cantharidin resistance.

Characterization and detection of maize-associated pteridovirus (MaPV), infecting maize (Zea mays) in the Arusha region of Tanzania

Maize lethal necrosis disease (MLND) is currently threatening maize production in a large area of East Africa. Synergistic infections of Maize chlorotic mottle virus (MCMV) and members of the Potyviridae family are known to elicit MLND. Metaviromics studies of plant viruses have allowed for the discovery of novel viral diversity, with a number of these viruses having recently been shown to co-occur with MCMV and Potyviruses. However, their contribution to the expression of disease symptoms still requires extensive research. In a survey for viruses associated with maize in Tanzania, 35 samples were sequenced using an RNA-tag-seq metaviromics approach. Bioinformatic analysis of assembled reads yielded two contigs (5.8 and 2.7 kbp) that shared sequence homology with RNA1 and RNA2 of the dsRNA Japanese holly fern mottle virus (JHFMoV) (genus: Pteridovirus) from a single sample collected in the Arusha region. RNA1 encodes for a polyprotein product containing putative viral methyltransferase, helicase and polymerase domains. RNA2 contains three open reading frames (ORFs), one of which encodes for a putative movement protein, while the remaining two encode for putative products with no known function. The tentative name Maize-associated pteridovirus (MaPV) has been proposed for the virus in this study.

Site-specific manipulation of Arabidopsis loci using CRISPR-Cas9 SunTag systems

Understanding genomic functions requires site-specific manipulation of loci via efficient protein effector targeting systems. However, few approaches for targeted manipulation of the epigenome are available in plants. Here, we adapt the dCas9-SunTag system to engineer targeted gene activation and DNA methylation in Arabidopsis. We demonstrate that a dCas9-SunTag system utilizing the transcriptional activator VP64 drives robust and specific activation of several loci, including protein coding genes and transposable elements, in diverse chromatin contexts. In addition, we present a CRISPR-based methylation targeting system for plants, utilizing a SunTag system with the catalytic domain of the Nicotiana tabacum DRM methyltransferase, which efficiently targets DNA methylation to specific loci, including the FWA promoter, triggering a developmental phenotype, and the SUPERMAN promoter. These SunTag systems represent valuable tools for the site-specific manipulation of plant epigenomes.

The ezh2(sa1199) mutant zebrafish display no distinct phenotype.

Polycomb group (PcG) proteins are essential regulators of epigenetic gene silencing and development. The PcG protein enhancer of zeste homolog 2 (Ezh2) is a key component of the Polycomb Repressive Complex 2 and is responsible for placing the histone H3 lysine 27 trimethylation (H3K27me3) repressive mark on the genome through its methyltransferase domain. Ezh2 is highly conserved in vertebrates. We studied the role of ezh2 during development of zebrafish with the use of a mutant allele (ezh2(sa1199), R18STOP), which has a stop mutation in the second exon of the ezh2 gene. Two versions of the same line were used during this study. The first and original version of zygotic ezh2(sa1199) mutants unexpectedly retained ezh2 expression in brain, gut, branchial arches, and eyes at 3 days post-fertilization (dpf), as revealed by in-situ hybridization. Moreover, the expression pattern in homozygous mutants was identical to that of wild types, indicating that mutant ezh2 mRNA is not subject to nonsense mediated decay (NMD) as predicted. Both wild type and ezh2 mutant embryos presented edemas at 2 and 3 dpf. The line was renewed by selective breeding to counter select the non-specific phenotypes and survival was assessed. In contrast to earlier studies on ezh2 mutant zebrafish, ezh2(sa1199) mutants survived until adulthood. Interestingly, the ezh2 mRNA and Ezh2 protein were present during adulthood (70 dpf) in both wild type and ezh2(sa1199) mutant zebrafish. We conclude that the ezh2(sa1199) allele does not exhibit an ezh2 loss-of-function phenotype.

Comparative Analysis of NS5 Protein for Tick Borne Encephalitis Virus Strains in three Virus Subtypes

Non-structural protein 5 (NS5) of tick-borne encephalitis virus is an enzyme which is responsible for a copying of viral RNA, and it has a strong structural similarity to RNA polymerases of another RNA virus families. The strains of the virus are separated into three subtypes, which differ by specific mutations in virus proteins, including NS5 protein. The methods of structural bioinformatics allow to construct a model of NS5 protein for several strains of the virus.The paper presents the comparative analysis of sequences and structures of NS5 protein, for three subtypes of the tick-borne encephalitis virus. The segments of protein were identified where the highest difference between subtypes and within subtypes is observed. These segments, where most of the mutations are accumulated, are located in methyltransferase domain, in the inter-domain interface, and in the three subdomains of polymerase domain. The association between the locations of mutations in NS5 protein and the flexibility of a protein backbone was observed using normal mode analysis. Namely, the most important mutations are located in the parts of protein where the amplitude of synchronous oscillations estimated using normal mode analysis is the highest: in the second zinc binding pocket within polymerase domain, in the N-terminal extension within inter-domain interface, and around an active site of methyltransferase domain.

M58 - FUNCTIONAL ANALYSIS OF THE SCHIZOPHRENIA ASSOCIATED GENE AS3MT IN SH-SY5Y NEUROBLASTOMA CELLS

Background Schizophrenia is a neuropsychiatric disorder with a prevalence of 1%, characterised by episodes of psychosis and an alteration in cognitive function. The aetiology of Schizophrenia is still largely unknown but evidence suggests an underlying neurodevelopmental aspect despite onset occurring in adulthood as well as a considerable genetic burden. Recently 108 genomic loci have robustly been associated with Schizophrenia. However, of these identified genes, very few have been characterised for their role in brain development. Arsenite Methyltransferase (AS3MT) is located in the10q24.32 GWAS locus, which is the most statistically significant locus outside the Major Histocompatibility Complex (MHC) region associated with schizophrenia. Furthermore, both mQTLs and expression quantitative trait loci (eQTL) map to this gene and a recent publication has identified an alternative splice variant which is increased in schizophrenia brain. AS3MT encodes for a methyltransferase involved in arsenic metabolism, however the role of AS3MT in brain development has not been explored. Methods CRISPR-Cas9 technology was used to create a knockout cell line of AS3MT in SH-SY5Y neuroblastoma cells. Two guide RNAs (gRNA) were designed, one to cut in exon 4 and one in exon 6 of the AS3MT gene, where the methyltransferase domain is located. These guides were cloned into two expression vectors which express the gRNA, Cas9 enzyme and either EGFP or mCherry. These vectors were transfected into P11 SH-SY5Y (neuroblastoma) cell line and screened for double fluorescence and sorted into single cells by Fluorescent Activated Cell Sorting after 24 hours. These cells were allowed to clonally expand for 3 weeks before DNA was extracted for genotyping by PCR. Protein expression of AS3MT was measured by western blotting, gene expression by RT-PCR and DNA methylation by the Ilumina EPIC array. Immunocytochemistry techniques will be used to identify any alterations in cell morphology. Results Of the cells transfected with EGPF and mCherry expressing CRISPR-Cas9 vectors 15.5% of the single sorted cells expressed both EGPF and mCherry and were sorted into 96 well plates. Following a week of clonal expansion approximately 38% of wells contained colonies, where 41% un-transfected control sorted cells had colonies. Following genotyping 27.8% (5/18) were identified as heterozygous mutants, 16.7% (3/18) were homozygous mutants and 55.6% (10/18) were wild type. Discussion We have developed a robust protocol for creating deletion mutants using CRISPR-Cas9 technology and a functional domain of AS3MT has been deleted in the neuroblastoma cell line SH-SY5Y using CRISPR-Cas9 technology, further work is ongoing to characterise the molecular consequences of this mutation.

Structural basis of 7SK RNA 5'-γ-phosphate methylation and retention by MePCE.

Among RNA 5′-cap structures, γ-phosphate monomethylation is unique to a small subset of noncoding RNAs, 7SK and U6 in humans. 7SK is capped by methylphosphate capping enzyme (MePCE), which has a second non-enzymatic role as a core component of the 7SK RNP that is an essential regulator of RNA transcription. We report 2.0 and 2.1 Å X-ray crystal structures of human MePCE methyltransferase domain bound to S-adenosylhomocysteine (SAH) and uncapped or capped 7SK substrates, respectively. 7SK recognition is achieved by protein contacts to a 5′ hairpin-single-stranded RNA region, explaining MePCE specificity for 7SK and U6. The structures reveal SAH and product RNA in a near-transition state geometry. Surprisingly, binding experiments show that MePCE has higher affinity for capped vs uncapped 7SK, with kinetic data supporting a slow product release model. This work reveals the molecular mechanism of methyl transfer and 7SK retention by MePCE for subsequent assembly of 7SK RNP.

Structural Basis of DNMT1 and DNMT3A-Mediated DNA Methylation.

DNA methylation, one of the major epigenetic mechanisms, plays critical roles in regulating gene expression, genomic stability and cell lineage commitment. The establishment and maintenance of DNA methylation in mammals is achieved by two groups of DNA methyltransferases (DNMTs): DNMT3A and DNMT3B, which are responsible for installing DNA methylation patterns during gametogenesis and early embryogenesis, and DNMT1, which is essential for propagating DNA methylation patterns during replication. Both groups of DNMTs are multi-domain proteins, containing a large N-terminal regulatory region in addition to the C-terminal methyltransferase domain. Recent structure-function investigations of the individual domains or large fragments of DNMT1 and DNMT3A have revealed the molecular basis for their substrate recognition and specificity, intramolecular domain-domain interactions, as well as their crosstalk with other epigenetic mechanisms. These studies highlight a multifaceted regulation for both DNMT1 and DNMT3A/3B, which is essential for the precise establishment and maintenance of lineage-specific DNA methylation patterns in cells. This review summarizes current understanding of the structure and mechanism of DNMT1 and DNMT3A-mediated DNA methylation, with emphasis on the functional cooperation between the methyltransferase and regulatory domains.

Stuffed Methyltransferase Catalyzes the Penultimate Step of Pyochelin Biosynthesis.

Nonribosomal peptide synthetases use tailoring domains to incorporate chemical diversity into the final natural product. A structurally unique set of tailoring domains are found to be stuffed within adenylation domains and have only recently begun to be characterized. PchF is the NRPS termination module in pyochelin biosynthesis and includes a stuffed methyltransferase domain responsible for S-adenosylmethionine (AdoMet)-dependent N-methylation. Recent studies of stuffed methyltransferase domains propose a model in which methylation occurs on amino acids after adenylation and thiolation rather than after condensation to the nascent peptide chain. Herein, we characterize the adenylation and stuffed methyltransferase didomain of PchF through the synthesis and use of substrate analogues, steady-state kinetics, and onium chalcogen effects. We provide evidence that methylation occurs through an SN2 reaction after thiolation, condensation, cyclization, and reduction of the module substrate cysteine and is the penultimate step in pyochelin biosynthesis.

The Structure of the Bifunctional Everninomicin Biosynthetic Enzyme EvdMO1 Suggests Independent Activity of the Fused Methyltransferase-Oxidase Domains.

Members of the orthosomycin family of natural products are decorated polysaccharides with potent antibiotic activity and complex biosynthetic pathways. The defining feature of the orthosomycins is an orthoester linkage between carbohydrate moieties that is necessary for antibiotic activity and is likely formed by a family of conserved oxygenases. Everninomicins are octasaccharide orthosomycins produced by Micromonospora carbonacea that have two orthoester linkages and a methylenedioxy bridge, three features whose formation logically requires oxidative chemistry. Correspondingly, the evd gene cluster encoding everninomicin D encodes two monofunctional nonheme iron, α-ketoglutarate-dependent oxygenases and one bifunctional enzyme with an N-terminal methyltransferase domain and a C-terminal oxygenase domain. To investigate whether the activities of these domains are linked in the bifunctional enzyme EvdMO1, we determined the structure of the N-terminal methyltransferase domain to 1.1 Å and that of the full-length protein to 3.35 Å resolution. Both domains of EvdMO1 adopt the canonical folds of their respective superfamilies and are connected by a short linker. Each domain's active site is oriented such that it faces away from the other domain, and there is no evidence of a channel connecting the two. Our results support EvdMO1 working as a bifunctional enzyme with independent catalytic activities.

Structural and Functional Studies of a gem-Dimethylating Methyltransferase from a trans-Acyltransferase Assembly Line.

The methyl substituents in products of trans-acyltransferase assembly lines are usually incorporated by S-adenosyl-methionine (SAM)-dependent methyltransferase (MT) domains. The gem-dimethyl moieties within the polyketide disorazol are installed through the iterative action of an MT in the third module of its assembly line. The 1.75-Å-resolution crystal structure of this MT helps elucidate how it catalyzes the addition of two methyl groups. Activity assays of point mutants on β-ketoacyl chains linked to an acyl carrier protein and N-acetylcysteamine provide additional insights into the roles of active site residues. The replacement of an alanine with a phenylalanine at an apparent gatekeeping position resulted in more monomethylation than dimethylation. MTs may form an interface with ketoreductases (KRs) and even mediate the docking of trans-acyltransferase assembly line polypeptides through this association.

Sclerotinia minor Endornavirus 1, a Novel Pathogenicity Debilitation-Associated Mycovirus with a Wide Spectrum of Horizontal Transmissibility.

Sclerotinia minor is a phytopathogenic fungus causing sclerotinia blight on many economically important crops. Here, we have characterized the biological and molecular properties of a novel endornavirus, Sclerotinia minor endornavirus 1 (SmEV1), isolated from the hypovirulent strain LC22 of S. minor. The genome of SmEV1 is 12,626 bp long with a single, large open reading frame (ORF), coding for a putative protein of 4020 amino acids. The putative protein contains cysteine-rich region (CRR), viral methyltransferase (MTR), putative DEXDc, viral helicase (Hel), and RNA-dependent RNA polymerase (RdRp) domains. The putative protein and the conserved domains are phylogenetically related to endornaviruses. SmEV1 does not contain a site-specific nick characteristic of most previously described endornaviruses. Hypovirulence and associated traits of strain LC22 and SmEV1 were readily cotransmitted horizontally via hyphal contact to isolates of different vegetative compatibility groups of S. minor. Additionally, SmEV1 in strain LC22 was found capable of being transmitted vertically through sclerotia. Furthermore, mycelium fragments of hypovirulent strain LC22 have a protective activity against attack by S. minor. Taken together, we concluded that SmEV1 is a novel hypovirulence-associated mycovirus with a wide spectrum of transmissibility, and has potential for biological control (virocontrol) of diseases caused by S. minor.