Proline/alanine-rich sequence (PAS) polypeptides as an alternative to PEG precipitants for protein crystallization.

Proline/alanine-rich sequence (PAS) polypeptides represent a novel class of biosynthetic polymers comprising repetitive sequences of the small proteinogenic amino acids L-proline, L-alanine and/or L-serine. PAS polymers are strongly hydrophilic and highly soluble in water, where they exhibit a natively disordered conformation without any detectable secondary or tertiary structure, similar to polyethylene glycol (PEG), which constitutes the most widely applied precipitant for protein crystallization to date. To investigate the potential of PAS polymers for structural studies by X-ray crystallography, two proteins that were successfully crystallized using PEG in the past, hen egg-white lysozyme and the Fragaria × ananassa O-methyltransferase, were subjected to crystallization screens with a 200-residue PAS polypeptide. The PAS polymer was applied as a precipitant using a vapor-diffusion setup that allowed individual optimization of the precipitant concentration in the droplet in the reservoir. As a result, crystals of both proteins showing high diffraction quality were obtained using the PAS precipitant. The genetic definition and precise macromolecular composition of PAS polymers, both in sequence and in length, distinguish them from all natural and synthetic polymers that have been utilized for protein crystallization so far, including PEG, and facilitate their adaptation for future applications. Thus, PAS polymers offer potential as novel precipitants for biomolecular crystallography.

Characterization of a Flavonoid 3'/5'/7-O-Methyltransferase from Citrus reticulata and Evaluation of the In Vitro Cytotoxicity of Its Methylated Products.

O-methylation of flavonoids is an important modification reaction that occurs in plants. O-methylation contributes to the structural diversity of flavonoids, which have several biological and pharmacological functions. In this study, an O-methyltransferase gene (CrOMT2) was isolated from the fruit peel of Citrus reticulata, which encoding a multifunctional O-methyltransferase and could effectively catalyze the methylation of 3'-, 5'-, and 7-OH of flavonoids with vicinal hydroxyl substitutions. Substrate preference assays indicated that this recombinant enzyme favored polymethoxylated flavones (PMF)-type substrates in vitro, thereby providing biochemical evidence for the potential role of the enzyme in plants. Additionally, the cytotoxicity of the methylated products from the enzymatic catalytic reaction was evaluated in vitro using human gastric cell lines SGC-7901 and BGC-823. The results showed that the in vitro cytotoxicity of the flavonoids with the unsaturated C2-C3 bond was increased after being methylated at position 3'. These combined results provide biochemical insight regarding CrOMT2 in vitro and indicate the in vitro cytotoxicity of the products methylated by its catalytic reaction.

Two dedicated class C radical S-adenosylmethionine methyltransferases concertedly catalyse the synthesis of 7,8-dimethylmenaquinone.

Dimethylmenaquinone (DMMK), a prevalent menaquinone (MK) derivative of uncertain function, is characteristic for members of the class Coriobacteriia. Such bacteria are frequently present in intestinal microbiomes and comprise several pathogenic species. The coriobacterial model organism Adlercreutzia equolifaciens was used to investigate the enzymology of DMMK biosynthesis. A HemN-like class C radical S-adenosylmethionine methyltransferase (MenK2) from A. equolifaciens was produced in Wolinella succinogenes or Escherichia coli cells and found to methylate MK specifically at position C-7. In combination with a previously described MK methyltransferase (MqnK/MenK) dedicated to MK methylation at C-8, 7,8-DMMK6 was produced in W. succinogenes. The position of the two methyl groups was confirmed by two-dimensional NMR and midpoint redox potentials of 7-MMK6, 8-MMK6 and 7,8-DMMK6 were determined by cyclic voltammetry. A phylogenetic tree of MenK, MenK2 and HemN proteins revealed a Coriobacteriia-specific MenK2 clade. Using chimeric A. equolifaciens MenK/MenK2 proteins produced in E. coli it was shown that the combined linker and HemN domains determined the site-specificity of methylation. The results suggest that the use of MenK2 as a biomarker allows predicting the ability of DMMK synthesis in microbial species.

Mycofactocin biosynthesis: modification of the peptide MftA by the radical S-adenosylmethionine protein MftC.

Mycofactocin is a putative, peptide derived, cofactor that is associated primarily with the Mycobacterium genera including the pathogen M. tuberculosis. The pathway consists of the three genes mftA, mftB, and mftC that encode for the peptide substrate, peptide chaperone, and a radical S-adenosylmethionine protein (RS), respectively. Here, we show that the MftB acts as a peptide chaperone, binding MftA with a submicromolar KD (~ 100 nm) and MftC with a low micromolar KD (~ 2 µm). Moreover, we demonstrate that MftC is a radical S-adenosylmethionine (SAM) enzyme. Finally, we show that MftC catalyzes the oxidative decarboxylation of the peptide MftA.

Crystal structure of O-methyltransferase CalO6 from the calicheamicin biosynthetic pathway: a case of challenging structure determination at low resolution.

BACKGROUND: Calicheamicins (CAL) are enedyine natural products with potent antibiotic and cytotoxic activity, used in anticancer therapy. The O-methyltransferase CalO6 is proposed to catalyze methylation of the hydroxyl moiety at the C2 position of the orsellinic acid group of CAL. RESULTS: Crystals of CalO6 diffracted non-isotropically, with the usable data extending to 3.4 Å. While no single method of crystal structure determination yielded a structure of CalO6, we were able to determine its structure by using molecular replacement-guided single wavelength anomalous dispersion by using diffraction data from native crystals of CalO6 and a highly non-isomorphous mercury derivative. The structure of CalO6 reveals the methyltransferase fold and dimeric organization characteristic of small molecule O-methyltransferases involved in secondary metabolism in bacteria and plants. Uncommonly, CalO6 was crystallized in the absence of S-adenosylmethionine (SAM; the methyl donor) or S-adenosylhomocysteine (SAH; its product). CONCLUSIONS: Likely as a consequence of the dynamic nature of CalO6 in the absence of its cofactor, the central region of CalO6, which forms a helical lid-like structure near the active site in CalO6 and similar enzymes, is not observed in the electron density. We propose that this region controls the entry of SAM into and the exit of SAH from the active site of CalO6 and shapes the active site for substrate binding and catalysis.

O-Methyltransferases involved in biphenyl and dibenzofuran biosynthesis.

Biphenyls and dibenzofurans are the phytoalexins of the Malinae involving apple and pear. Biosynthesis of the defence compounds includes two O-methylation reactions. cDNAs encoding the O-methyltransferase (OMT) enzymes were isolated from rowan (Sorbus aucuparia) cell cultures after treatment with an elicitor preparation from the scab-causing fungus, Venturia inaequalis. The preferred substrate for SaOMT1 was 3,5-dihydroxybiphenyl, supplied by the first pathway-specific enzyme, biphenyl synthase (BIS). 3,5-Dihydroxybiphenyl underwent a single methylation reaction in the presence of S-adenosyl-l-methionine (SAM). The second enzyme, SaOMT2, exhibited its highest affinity for noraucuparin, however the turnover rate was greater with 5-hydroxyferulic acid. Both substrates were only methylated at the meta-positioned hydroxyl group. The substrate specificities of the OMTs and the regiospecificities of their reactions were rationalized by homology modeling and substrate docking. Interaction of the substrates with SAM also took place at a position other than the sulfur group. Expression of SaOMT1, SaOMT2 and SaBIS3 was transiently induced in rowan cell cultures by the addition of the fungal elicitor. While the immediate SaOMT1 products were not detectable in elicitor-treated cell cultures, noraucuparin and noreriobofuran accumulated transiently, followed by increasing levels of the SaOMT2 products aucuparin and eriobofuran. SaOMT1, SaOMT2 and SaBIS3 were N- and C-terminally fused with the super cyan fluorescent protein and a modified yellow fluorescent protein, respectively. All the fluorescent reporter fusions were localized to the cytoplasm of Nicotiana benthamiana leaf epidermis cells. A revised biosynthetic pathway of biphenyls and dibenzofurans in the Malinae is presented.

A LCMT1-PME-1 methylation equilibrium controls mitotic spindle size.

Leucine carboxyl methyltransferase-1 (LCMT1) and protein phosphatase methylesterase-1 (PME-1) are essential enzymes that regulate the methylation of the protein phosphatase 2A catalytic subunit (PP2AC). LCMT1 and PME-1 have been linked to the regulation of cell growth and proliferation, but the underlying mechanisms have remained elusive. We show here an important role for an LCMT1-PME-1 methylation equilibrium in controlling mitotic spindle size. Depletion of LCMT1 or overexpression of PME-1 led to long spindles. In contrast, depletion of PME-1, pharmacological inhibition of PME-1 or overexpression of LCMT1 led to short spindles. Furthermore, perturbation of the LCMT1-PME-1 methylation equilibrium led to mitotic arrest, spindle assembly checkpoint activation, defective cell divisions, induction of apoptosis and reduced cell viability. Thus, we propose that the LCMT1-PME-1 methylation equilibrium is critical for regulating mitotic spindle size and thereby proper cell division.

Crystallization and preliminary X-ray crystallographic studies of CrArsM, an arsenic(III) S-adenosylmethionine methyltransferase from Chlamydomonas reinhardtii.

Arsenic is one the most toxic environmental substances. Arsenic is ubiquitous in water, soil and food, and ranks first on the Environmental Protection Agency's Superfund Priority List of Hazardous Substances. Arsenic(III) S-adenosylmethionine methyltransferases (AS3MT in animals and ArsM in microbes) are key enzymes of arsenic biotransformation, catalyzing the methylation of inorganic arsenite to give methyl, dimethyl and trimethyl products. Arsenic methyltransferases are found in members of every kingdom from bacteria to humans (EC 2.1.1.137). In the human liver, hAS3MT converts inorganic arsenic into more toxic and carcinogenic forms. CrArsM, an ortholog of hAS3MT from the eukaryotic green alga Chlamydomonas reinhardtii, was purified by chemically synthesizing the gene and expressing it in Escherichia coli. Synthetic purified CrArsM was crystallized in an unliganded form. Crystals were obtained by the hanging-drop vapor-diffusion method. The crystals belonged to space group R3:H, with unit-cell parameters a = b = 157.8, c = 95.4 Å, γ = 120° and two molecules in the asymmetric unit. Complete data sets were collected and processed to a resolution of 2.40 Å.

The BioC O-methyltransferase catalyzes methyl esterification of malonyl-acyl carrier protein, an essential step in biotin synthesis.

Recent work implicated the Escherichia coli BioC protein as the initiator of the synthetic pathway that forms the pimeloyl moiety of biotin (Lin, S., Hanson, R. E., and Cronan, J. E. (2010) Nat. Chem. Biol. 6, 682-688). BioC was believed to be an O-methyltransferase that methylated the free carboxyl of either malonyl-CoA or malonyl-acyl carrier protein based on the ability of O-methylated (but not unmethylated) precursors to bypass the BioC requirement for biotin synthesis both in vivo and in vitro. However, only indirect proof of the hypothesized enzymatic activity was obtained because the activities of the available BioC preparations were too low for direct enzymatic assay. Because E. coli BioC protein was extremely recalcitrant to purification in an active form, BioC homologues of other bacteria were tested. We report that the native form of Bacillus cereus ATCC10987 BioC functionally replaced E. coli BioC in vivo, and the protein could be expressed in soluble form and purified to homogeneity. In disagreement with prior scenarios that favored malonyl-CoA as the methyl acceptor, malonyl-acyl carrier protein was a far better acceptor of methyl groups from S-adenosyl-L-methionine than was malonyl-CoA. BioC was specific for the malonyl moiety and was inhibited by S-adenosyl-L-homocysteine and sinefungin. High level expression of B. cereus BioC in E. coli blocked cell growth and fatty acid synthesis.

Structural and functional dissection of aminocoumarin antibiotic biosynthesis: a review.

Aminocoumarin antibiotics are natural products of soil-dwelling bacteria called Streptomycetes. They are potent inhibitors of DNA gyrase, an essential bacterial enzyme and validated drug target, and thus have attracted considerable interest as potential templates for drug development. To date, aminocoumarins have not seen widespread clinical application on account of their poor pharmacological properties. Through studying the structures and mechanisms of enzymes from their biosynthetic pathways we will be better informed to redesign these compounds through rational pathway engineering. Novobiocin, the simplest compound, requires at least seventeen gene products to convert primary metabolites into the mature antibiotic. We have solved the crystal structures of four diverse biosynthetic enzymes from the novobiocin pathway, and used these as three-dimensional frameworks for the interpretation of functional and mechanistic data, and to speculate about how they might have evolved. The structure determinations have ranged from the routine to the challenging, necessitating a variety of different approaches.

Methylation of a phosphatase specifies dephosphorylation and degradation of activated brassinosteroid receptors.

Internalization of cell surface receptors, followed by either recycling back to the plasma membrane or degradation, is crucial for receptor homeostasis and signaling. The plant brassinosteroid (BR) receptor, BRASSINOSTEROID INSENSITIVE 1 (BRI1), undergoes constitutive cycling between the plasma membrane and the internal membranes. We show that protein phosphatase 2A (PP2A) dephosphorylated BRI1 and that Arabidopsis thaliana rcn1, a mutant for a PP2A subunit, caused an increase in BRI1 abundance and BR signaling. We report the identification, in A. thaliana, of a suppressor of bri1, sbi1, which caused selective accumulation of BR-activated BRI1, but not the BR co-receptor BAK1 (BRI1-ASSOCIATED KINASE 1), in the membranous compartment. SBI1 mRNA was induced by BRs, and SBI1 encodes a leucine carboxylmethyltransferase (LCMT) that methylated PP2A and controlled its membrane-associated subcellular localization. We propose that BRs increase production of SBI1, which methylates PP2A, thus facilitating its association with activated BRI1. This leads to receptor dephosphorylation and degradation, and thus to the termination of BR signaling.

The structural basis for tight control of PP2A methylation and function by LCMT-1.

Proper formation of protein phosphatase 2A (PP2A) holoenzymes is essential for the fitness of all eukaryotic cells. Carboxyl methylation of the PP2A catalytic subunit plays a critical role in regulating holoenzyme assembly; methylation is catalyzed by PP2A-specific methyltransferase LCMT-1, an enzyme required for cell survival. We determined crystal structures of human LCMT-1 in isolation and in complex with PP2A stabilized by a cofactor mimic. The structures show that the LCMT-1 active-site pocket recognizes the carboxyl terminus of PP2A, and, interestingly, the PP2A active site makes extensive contacts to LCMT-1. We demonstrated that activation of the PP2A active site stimulates methylation, suggesting a mechanism for efficient conversion of activated PP2A into substrate-specific holoenzymes, thus minimizing unregulated phosphatase activity or formation of inactive holoenzymes. A dominant-negative LCMT-1 mutant attenuates the cell cycle without causing cell death, likely by inhibiting uncontrolled phosphatase activity. Our studies suggested mechanisms of LCMT-1 in tight control of PP2A function, important for the cell cycle and cell survival.

The structure of human leucine carboxyl methyltransferase 1 that regulates protein phosphatase PP2A.

Leucine carboxyl methyltransferase 1 (LCMT1) methylates the terminal carboxyl group of the leucine 309 residue of human protein phosphatase 2A (PP2A). PP2A, a key regulator of many cellular processes, has recently generated additional interest as a potential cancer-therapeutic target. The status of PP2A methylation impacts upon the selection of the regulatory subunit by the PP2A core enzyme, thus directing its activity and subcellular localization. An X-ray crystal structure of human LCMT1 protein in complex with the cofactor S-adenosylmethionine (AdoMet) has been solved to a resolution of 2 Å. The structure enables the postulation of a mode of interaction with protein phosphatase PP2A and provides a platform for further functional studies of the regulation of methylation of PP2A.

The crystal structure of the novobiocin biosynthetic enzyme NovP: the first representative structure for the TylF O-methyltransferase superfamily.

NovP is an S-adenosyl-l-methionine-dependent O-methyltransferase that catalyzes the penultimate step in the biosynthesis of the aminocoumarin antibiotic novobiocin. Specifically, it methylates at 4-OH of the noviose moiety, and the resultant methoxy group is important for the potency of the mature antibiotic: previous crystallographic studies have shown that this group interacts directly with the target enzyme DNA gyrase, which is a validated drug target. We have determined the high-resolution crystal structure of NovP from Streptomyces spheroides as a binary complex with its desmethylated cosubstrate S-adenosyl-l-homocysteine. The structure displays a typical class I methyltransferase fold, in addition to motifs that are consistent with a divalent-metal-dependent mechanism. This is the first representative structure of a methyltransferase from the TylF superfamily, which includes a number of enzymes implicated in the biosynthesis of antibiotics and other therapeutics. The NovP structure reveals a number of distinctive structural features that, based on sequence conservation, are likely to be characteristic of the superfamily. These include a helical 'lid' region that gates access to the cosubstrate binding pocket and an active center that contains a 3-Asp putative metal binding site. A further conserved Asp likely acts as the general base that initiates the reaction by deprotonating the 4-OH group of the noviose unit. Using in silico docking, we have generated models of the enzyme-substrate complex that are consistent with the proposed mechanism. Furthermore, these models suggest that NovP is unlikely to tolerate significant modifications at the noviose moiety, but could show increasing substrate promiscuity as a function of the distance of the modification from the methylation site. These observations could inform future attempts to utilize NovP for methylating a range of glycosylated compounds.

Molecular basis of substrate promiscuity for the SAM-dependent O-methyltransferase NcsB1, involved in the biosynthesis of the enediyne antitumor antibiotic neocarzinostatin.

The small molecule component of chromoprotein enediyne antitumor antibiotics is biosynthesized through a convergent route, incorporating amino acid, polyketide, and carbohydrate building blocks around a central enediyne hydrocarbon core. The naphthoic acid moiety of the enediyne neocarzinostatin plays key roles in the biological activity of the natural product by interacting with both the carrier protein and duplex DNA at the site of action. We have previously described the in vitro characterization of an S-adenosylmethionine-dependent O-methyltransferase (NcsB1) in the neocarzinostatin biosynthetic pathway [Luo, Y., Lin, S., Zhang, J., Cooke, H. A., Bruner, S. D., and Shen, B. (2008) J. Biol. Chem. 283, 14694-14702]. Here we provide a structural basis for NcsB1 activity, illustrating that the enzyme shares an overall architecture with a large family of S-adenosylmethionine-dependent proteins. In addition, NcsB1 represents the first enzyme to be structurally characterized in the biosynthetic pathway of neocarzinostatin. By cocrystallizing the enzyme with various combinations of the cofactor and substrate analogues, details of the active site structure have been established. Changes in subdomain orientation were observed via comparison of structures in the presence and absence of substrate, suggesting that reorientation of the enzyme is involved in binding of the substrate. In addition, residues important for substrate discrimination were predicted and probed through site-directed mutagenesis and in vitro biochemical characterization.

A novel cation-dependent O-methyltransferase involved in anthocyanin methylation in grapevine.

Anthocyanins are major pigments in colored grape (Vitis vinifera) berries, and most of them are monomethoxylated or dimethoxylated. We report here the functional characterization of an anthocyanin O-methyltransferase (AOMT) from grapevine. The expression pattern in two cultivars with different anthocyanin methylation profiles (Syrah and Nebbiolo) showed a peak at start ripening (véraison), when the concentrations of all methylated anthocyanins begin to increase. The purified recombinant AOMT protein was active on both anthocyanins and flavonols in vitro, with K(m) in the micromolar range, and was dependent on divalent cations for activity. AOMT showed a preference for 3',5' methylation when a 3',4',5' hydroxylated anthocyanin substrate was tested. In order to assess its in planta activity, we performed transient expression of AOMT in tobacco (Nicotiana benthamiana) leaves expressing the Production of Anthocyanin Pigment1 (PAP1) transcription factor from Arabidopsis (Arabidopsis thaliana). PAP1 expression in leaves induced the accumulation of the nonmethylated anthocyanin delphinidin 3-rutinoside. The coexpression of PAP1 and AOMT resulted in an accumulation of malvidin 3-rutinoside. We also showed that AOMT localized exclusively in the cytoplasm of tobacco leaf cells. These results demonstrate the ability of this enzyme to methylate anthocyanins both in vitro and in vivo, indicating that AOMT plays a major role in anthocyanin biosynthesis in grape berries.

Functional analysis of MycE and MycF, two O-methyltransferases involved in the biosynthesis of mycinamicin macrolide antibiotics.

Mg motors: We characterized the in vitro function of MycE and MycF, two O-methyltransferases involved in the biosynthesis of mycinamicin antibiotics. Each enzyme was confirmed to be an S-adenosyl-L-methionine (SAM)-dependent deoxysugar methyltransferase. Their optimal activities require the presence of Mg(2+). With the reconstituted in vitro assays, the order of mycinamicin VI-->III-->IV in the post-PKS (polyketide synthase) tailoring pathway of mycinamicin was unambiguously determined.

Altered regioselectivity of a poplar O-methyltransferase, POMT-7.

O-Methylated flavonoids are biosynthesized by regioselective flavonoid O-methyltransferases (OMTs), which may account for the limited number of naturally occurring flavonoids in nature. It was previously shown that poplar POMT-7 regioselectively methylates the 7-hydroxyl group of flavones, whereas rice ROMT-9 regioselectively methylates the 3'-hydroxyl group of the substrate. We co-expressed both OMT genes (POMT-7 and ROMT-9) in E. coli and carried out biotransformation experiments of some flavonoids with the transformed E. coli strain. Contrast to the predicted regioselectivity of both POMT-7 and ROMT-9, unexpected methylation reaction products, i.e. 3',4'-O-methylated flavonoids, in addition to the predicted ones, were obtained with luteolin (5,7,3',4'-tetrahydroxyflavone) and quercetin (3,5,7,3',4'-pentahydroxyflavone) as substrates. Reactions using the 3'-O-methyl derivative of luteolin and quercetin by POMT-7 revealed that the enzyme has altered its regioselectivity from the 7- to the 4'-hydroxyl groups. These results are discussed in terms of molecular modeling of POMT-7 in relation to its methyl donor.

Biochemical and structural analysis of substrate promiscuity in plant Mg2+-dependent O-methyltransferases.

Plant S-adenosyl-l-methionine-dependent class I natural product O-methyltransferases (OMTs), related to animal catechol OMTs, are dependent on bivalent cations and strictly specific for the meta position of aromatic vicinal dihydroxy groups. While the primary activity of these class I enzymes is methylation of caffeoyl coenzyme A OMTs, a distinct subset is able to methylate a wider range of substrates, characterized by the promiscuous phenylpropanoid and flavonoid OMT. The observed broad substrate specificity resides in two regions: the N-terminus and a variable insertion loop near the C-terminus, which displays the lowest degree of sequence conservation between the two subfamilies. Structural and biochemical data, based on site-directed mutagenesis and domain exchange between the two enzyme types, present evidence that only small topological changes among otherwise highly conserved 3-D structures are sufficient to differentiate between an enzymatic generalist and an enzymatic specialist in plant natural product methylation.

Molecular cloning and characterization of O-methyltransferases from the flower buds of Iris hollandica.

In plants, O-methyltransferases (OMTs) play an important role in methylation of secondary metabolites, especially flavonoids and other phenylpropanoids, and two cDNA clones, IhOMT1 and IhOMT2 (Iris hollandica OMT), encoding OMTs were successfully isolated from a cDNA library of flower buds of I. hollandica. IhOMT1 encodes an open reading frame (ORF) of 365 amino acids with calculated molecular mass of 40,193Da and isoelectric point (pI) of 5.54, while IhOMT2, which shares 31.5% amino acid sequence identity with IhOMT1, encodes 369 amino acids with calculated molecular mass of 40,385Da and pI of 5.50. In addition, the molecular masses of both recombinant IhOMT1 and IhOMT2 proteins were estimated to be about 40kDa by protein gel blot analysis. Characterization of the enzymatic properties using the recombinant IhOMT1 protein confirmed that IhOMT1 cDNA encodes a S-adenosyl-L-methionine (SAM)-dependent caffeic acid 3-OMT, which catalyzes the transfer of the methyl moiety from SAM to caffeic acid to form ferulic acid. Its optimum activity was observed at pH 7.5-8.0 and at 35 degrees C. This is the first report of the isolation and characterization of a COMT cDNA clone involved in the phenylpropanoid biosynthesis of Iridaceae plants. In contrast, IhOMT2 showed no activity in SAM-dependent assays for various phenylpropanoids.