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1.
Bong-Gyu Kim Dae Hwan Kim Su Hyun Sung Dong-Eun Kim Youhoon Chong Joong-Hoon Ahn 《Planta》2010,232(4):837-844
O-Methyltransferase (OMT) catalyzes the transfer of a methyl group from S-adenosyl methionine (SAM) to hydroxyl groups of methyl acceptors. Two OMTs, PaOMT2 and PaOMT3, from Picea abies showed 93.5% identity at the amino acid level. However, PaOMT3 catalyzed the reaction more efficiently than PaOMT2 with several
phenolic compounds, including quercetin and caffeoyl-CoA. To determine the critical amino acids for the different reactivity
of the two OMTs, site-directed mutagenesis was carried out. The amino acid proline at position 35 in PaOMT2 and leucine in
PaOMT3 is a critical amino acid for their reactivity. Molecular modeling showed that the sequential change triggered by Leu35
resulted in a change in the size of the substrate binding pocket, which could account for the different catalytic reactivity
of two OMTs. 相似文献
2.
Chiron H Drouet A Claudot AC Eckerskorn C Trost M Heller W Ernst D Sandermann H 《Plant molecular biology》2000,44(6):733-745
Formation of pinosylvin (PS) and pinosylvin 3-O-monomethyl ether (PSM), as well as the activities of stilbene synthase (STS) and S-adenosyl-l-methionine (SAM):pinosylvin O-methyltransferase (PMT), were induced strongly in needles of Scots pine seedlings upon ozone treatment, as well as in cell suspension cultures of Scots pine upon fungal elicitation. A SAM-dependent PMT protein was purified and partially characterised. A cDNA encoding PMT was isolated from an ozone-induced Scots pine cDNA library. Southern blot analysis of the genomic DNA suggested the presence of a gene family. The deduced protein sequence showed the typical highly conserved regions of O-methyltransferases (OMTs), and average identities of 20–56% to known OMTs. PMT expressed in Escherichia coli corresponded to that of purified PMT (40 kDa) from pine cell cultures. The recombinant enzyme catalysed the methylation of PS, caffeic acid, caffeoyl-CoA and quercetin. Several other substances, such as astringenin, resveratrol, 5-OH-ferulic acid, catechol and luteolin, were also methylated. Recombinant PMT thus had a relatively broad substrate specificity. Treatment of 7-year old Scots pine trees with ozone markedly increased the PMT mRNA level. Our results show that PMT represents a new SAM-dependent OMT for the methylation of stress-induced pinosylvin in Scots pine needles. 相似文献
3.
Two lower molecular mass OMT genes (ROMT-15 and -17) were cloned from rice and expressed in Escherichia coli as glutathione S-transferase fusion proteins. ROMT-15 and -17 metabolized caffeoyl-CoA, flavones and flavonols containing two vicinal hydroxyl
groups, although they exhibited different substrate specificities. The position of methylation in both luteolin and quercetin
was determined to be the 3′ hydroxyl group and myricetin and tricetin were methylated not only at 3′ but also at 5′ hydroxyl
groups. ROMT-15 and -17 are cation-dependent and mutation of the predicted metal binding sites resulted in the loss of the
enzyme activity, indicating that the metal ion has a critical role in the enzymatic methylation. 相似文献
4.
Bong-Gyu Kim Su Hyun Sung Youhoon Chong Yoongho Lim Joong-Hoon Ahn 《Journal of Plant Biology》2010,53(5):321-329
Flavonoids consist of a large family of compounds, which has been estimated to be more than 10,000 compounds. The structural
diversity of these compounds comes from different modification reactions. The O-methylation reaction is one of the most important modification reactions of flavonoids and the resulting O-methylated flavonoids have been shown to display new biological activities. The regioselective and substrate specific O-methylation is mediated by O-methyltranferases (OMTs). To date, 30 flavonoid OMTs (FOMTs) have been biochemically characterized from various plants. FOMTs
utilize common reaction mechanisms to transfer a methyl group to the hydroxyl group of the flavonoid. Phylogenetic tree analysis
along with biochemical characterization of FOMTs provides clues about their substrate specificity and regioselectivity. FOMTs
can be used for the production of O-methylated flavonoids that have a particular biological activity. 相似文献
5.
Jeong Ho Kim Young Min Cheon Bong -Gyu Kim Joong -Hoon Ahn 《Journal of Plant Biology》2008,51(2):97-101
The major flavonoids in rice leaves were analyzed via LC-MS/MS after their total flavonoid extracts were hydrolyzed. The most
abundant flavones were apigenin, luteolin, and tricetin. Of these, tricetin was methylated at its 3′ and 5′-hydroxyl group
to form tricin, which was probablyO-glycosylated. Both 3′-O-methylated luteolin and luteolin were found in theC-glycosylated form while apigenin wasC-glycosylated. We also cloned and characterizedOsFNS, which catalyzes the reaction from flavanone (naringenin) to flavone (apigenin). Analysis of the reaction product with recombinant
OsFNS showed that it indeed converts naringenin to apigenin. 相似文献
6.
Evolution of Novel O-methyltransferases from the Vanilla planifolia Caffeic Acid O-methyltransferase
Li HM Rotter D Hartman TG Pak FE Havkin-Frenkel D Belanger FC 《Plant molecular biology》2006,61(3):537-552
The biosynthesis of many plant secondary compounds involves the methylation of one or more hydroxyl groups, catalyzed by O-methyltransferases (OMTs). Here, we report the characterization of two OMTs, Van OMT-2 and Van OMT-3, from the orchid Vanilla planifolia Andrews. These enzymes catalyze the methylation of a single outer hydroxyl group in substrates possessing a 1,2,3-trihydroxybenzene
moiety, such as methyl gallate and myricetin. This is a substrate requirement not previously reported for any OMTs. Based
on sequence analysis these enzymes are most similar to caffeic acid O-methyltransferases (COMTs), but they have negligible activity with typical COMT substrates. Seven of 12 conserved substrate-binding
residues in COMTs are altered in Van OMT-2 and Van OMT-3. Phylogenetic analysis of the sequences suggests that Van OMT-2 and
Van OMT-3 evolved from the V. planifolia COMT. These V. planifolia OMTs are new instances of COMT-like enzymes with novel substrate preferences. 相似文献
7.
Kopycki JG Rauh D Chumanevich AA Neumann P Vogt T Stubbs MT 《Journal of molecular biology》2008,378(1):154-164
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. 相似文献
8.
L. L. Zavalova N. V. Antipova Yu. I. Fadeeva M. S. Pavlyukov N. V. Pletneva V. Z. Pletnev I. P. Baskova 《Russian Journal of Bioorganic Chemistry》2012,38(2):198-202
Based on the three-dimensional model of the bifunctional enzyme destabilase-lysozyme of the medicinal leech (mlDL) in complex
with trimer of N-acetylglucosamine (NAG)3 by site-directed mutagenesis method, the functional role of the group of amino acids (Glu14, Asp26, Ser29, Ser31, Lys38,
His92) in manifestation of lysozyme (glycosidase, muramidase) and isopeptidase activities has been investigated by site-directed
mutagenesis. The results obtained go well with hypothesis, that lysozyme active site of mlDL includes catalytic Glu14 and
Asp26 residues, and isopeptidase site functions as Ser/Lys catalytic dyad presented by catalytic residues Ser29 and Lys38.
Thus, among the invertebrate lysozymes, mlDL presents the first example of a bifunctional enzyme with identified position
of the isopeptidase active site and localization of the corresponding catalytic residues. 相似文献
9.
Kim NY Kim JH Lee YH Lee EJ Kim J Lim Y Chong Y Ahn JH 《Journal of microbiology and biotechnology》2007,17(12):1991-1995
Omicron-Methylation is a common substitution reaction found in microbes as well as in mammalians. Some of the Omicron-methyltransferases (OMTs) have broad substrate specificity and could be used to methylate various compounds. DnrK from Streptomyces peucetius encodes an anthracycline 4-Omicron-methyltransferase, which uses carminomycin as a substrate, and its crystal structure has been determined. Molecular docking experiments with DnrK using various flavonoids were successfully conducted, and some of the flavonoids such as apigenin and genistein were predicted to serve as substrates. Based on these results, Omicron-methylations of various flavonoids with the DnrK were successfully carried out. The methylation position was determined to be at the hydroxyl group of C7. Important amino acid residues for the enzymatic reaction of DnrK with apigenin could be identified using site-directed mutagenesis. Molecular docking could be useful to predict the substrate specificity ranges of other OMTs. 相似文献
10.
Minoru Yonezawa Masahiro Takahata Naoko Banzawa Nobuyuki Matsubara Yasuo Watanabe Hirokazu Narita 《Microbiology and immunology》1995,39(7):517-520
The functional contributions of amino acid residue Asp87 of Escherichia coli gyrase A protein (GyrA) was analyzed by site-directed mutagenesis. We generated a series of mutants, in which Asp87 of GyrA was changed to Ala, Val, Phe, Asn, Ser, and Lys. By genetic analysis of gyrA genes in a gyrA temperature-sensitive (Ts) background, it was shown that all these mutations caused the quinolone-resistance. These results indicate that the 87th amino acid of E. coli GyrA must have negative charge in expressing the phenotype of quinolone sensitivity. These findings also suggest that the carboxyl group of Asp87 may interact with quinolone drugs. 相似文献
11.
Widiez T Hartman TG Dudai N Yan Q Lawton M Havkin-Frenkel D Belanger FC 《Plant molecular biology》2011,76(6):475-488
Caffeoyl CoA O-methyltransferases (OMTs) have been characterized from numerous plant species and have been demonstrated to be involved in
lignin biosynthesis. Higher plant species are known to have additional caffeoyl CoA OMT-like genes, which have not been well
characterized. Here, we identified two new caffeoyl CoA OMT-like genes by screening a cDNA library from specialized hair cells
of pods of the orchid Vanilla planifolia. Characterization of the corresponding two enzymes, designated Vp-OMT4 and Vp-OMT5, revealed that in vitro both enzymes preferred
as a substrate the flavone tricetin, yet their sequences and phylogenetic relationships to other enzymes are distinct from
each other. Quantitative analysis of gene expression indicated a dramatic tissue-specific expression pattern for Vp-OMT4, which was highly expressed in the hair cells of the developing pod, the likely location of vanillin biosynthesis. Although
Vp-OMT4 had a lower activity with the proposed vanillin precursor, 3,4-dihydroxybenzaldehyde, than with tricetin, the tissue
specificity of expression suggests it may be a candidate for an enzyme involved in vanillin biosynthesis. In contrast, the
Vp-OMT5 gene was mainly expressed in leaf tissue and only marginally expressed in pod hair cells. Phylogenetic analysis suggests
Vp-OMT5 evolved from a cyanobacterial enzyme and it clustered within a clade in which the sequences from eukaryotic species
had predicted chloroplast transit peptides. Transient expression of a GFP-fusion in tobacco demonstrated that Vp-OMT5 was
localized in the plastids. This is the first flavonoid OMT demonstrated to be targeted to the plastids. 相似文献
12.
Prephenate dehydratase is a key regulatory enzyme in the phenylalanine-specific pathway of Corynebacterium glutamicum. PCR-based random mutagenesis and functional complementation were used to screen for m-fluorophenylalanine (mFP)-resistant mutants. Comparison of the amino acid sequence of the mutant prephenate dehydratases indicated that Ser-99 plays a role in the feedback regulation of the enzyme. When Ser-99 of the wild-type enzyme was replaced by Met, the specific activity of the mutant enzyme was 30% lower than that of the wild-type. The Ser99Met mutant was active in the presence of 50 M phenylalanine, whereas the wild-type enzyme was not. The functional roles of the eight conserved residues of prephenate dehydratase were investigated by site-directed mutagenesis. Glu64Asp substitution reduced enzyme activity by 15%, with a 4.5- and 1.7-fold increase in K
m and k
cat values, respectively. Replacement of Thr-183 by either Ala or Tyr resulted in a complete loss of enzyme activity. Substitution of Arg-184 with Leu resulted in a 50% decrease of enzyme activity. The specific activity for Phe185Tyr was more than 96% lower than that of the wild-type, and the K
m value was 26-fold higher. Alterations in the conserved Asp-76, Glu-89, His-115, and Arg-236 residues did not cause a significant change in the K
m and k
cat values. These results indicated that Glu-64, Thr-183, Arg-184, and Phe-185 residues might be involved in substrate binding and/or catalytic activity. 相似文献
13.
Chebotaev D. V. Gul'ko L. B. Veiko V. P. 《Russian Journal of Bioorganic Chemistry》2001,27(3):160-166
Genes for hybrid uridine phosphorylases (UPases) consisting of fragments of amino acid sequences of UPases from Escherichia
coliand Salmonella
typhimuriumwere constructed. Producing strains of the corresponding proteins were genetically engineered. Mutant forms of the E.
coliK-12 UPase were produced by site-directed mutagenesis. A comparative study of the enzyme properties of the mutant and hybrid forms of bacterial UPases was performed. It was shown that Asp27 rather than Asp5 and Asp29 residues of the E
coliUPase forms part of the active site of the protein. A scheme of the involvement of Asp27 in the binding of inorganic phosphate is proposed. 相似文献
14.
The partially purified O-methyltransferase (OMT) system of Chrysosplenium americanum was found to catalyse the stepwise O-methylation of quercetin to its mono-, di- and trimethyl derivatives. It also utilized the partially methylated flavonol intermediates to form the next higher order of O-methylated products; thus indicating the involvement of several OMTs. The latter were resolved by chromatofocusing into three distinct peaks of enzyme activity which focused at pI values 4.8, 5.4 and 5.7. The former enzyme O-methylated quercetin at the 3-position, whereas the latter two O-methylated 3, 7-di-O-methyl quercetagetin at the 3′- and 6-positions, respectively. None of the focused enzymes accepted caffeic acid, or other flavonoids such as kaempferol or luteolin, as substrates; thus indicating specificity towards flavonols with 3′, 4′- substitution. The three OMTs had similar MWs and the Km values for their substrates were of the same order of magnitude. The biochemical role of these novel enzymes is discussed in relation to the biosynthesis of polymethylated flavonols in this tissue. 相似文献
15.
Toshikazu Shiba Hiroshi Iwasaki Atsuo Nakata Hideo Shinagawa 《Molecular & general genetics : MGG》1990,224(2):169-176
Summary The mucAB operon carried on plasmid pKM101, which is an analogue of the umuDC operon of Escherichia coli, is involved in UV mutagenesis and mutagenesis induced by many chemicals. Mutagenesis dependent on either the umuDC or mucAB operon requires the function of the recA gene and is called SOS mutagenesis. By treating the cell with agents that damage DNA, RecA protein is activated by conversion into a form (RecA*) that mediates proteolytic cleavage of the LexA repressor and derepresses the SOS genes including mucAB. Since UmuD protein is proteolytically processed to an active form (UmuD*) in a RecA*-dependent fashion, and MucA shares extensive amino acid homology with UmuD, we examined whether MucA is similarly processed in the cell, using antiserum against a LacZ-MucA fusion protein. Like UmuD, MucA protein is indeed proteolytically processed in a RecA*-dependent fashion. In recA430 strains, MucAB but not UmuDC can mediate UV mutagenesis. However, MucA was not processed in the recA430 cells treated with mitomycin C. We constructed, by site-directed mutagenesis, several mutant mucA genes that encode MucA proteins with alterations in the amino acids flanking the putative cleavage site (Ala25-Gly26). MucA(Cys25) was processed and was as mutagenically active as wild-type MucA; MucA(Asp26) and MucA(Cys25,Asp26) were not processed, and were mutagenically inactive; MucA-(Thr25) was not processed, but was mutagenically as active as wild-type MucA. The mutant mucA gene that encoded the putative cleavage product of MucA was as active as mucA
+ in UV mutagenesis. These results raise the possibility that both the nascent MucA and the processed product are active in mutagenesis. 相似文献
16.
Bong Gyu Kim Yoon Jung Lee Younghee Park Yoongho Lim Joong-Hoon Ahn 《Journal of Plant Biology》2006,49(1):55-60
Enzymatic O-methylation, catalyzed by S-adenosyl-L-methionine (SAM)-dependent O-methyltranferases (OMTs), is a ubiquitous
reaction, occurring in almost all living organisms. Plant OMTs are involved in the methylation of secondary metabolites, including
phenylpropanoid and flavonoid compounds. Here, we used RT-PCR to isolate and characterizePOMT-2 fromPopulus deltoides. This OMT comprises a 1095-b open reading frame that encodes a 39.7-kDa protein. BLAST results showed 87% identities to an
OMT fromPrunus dulcis and a caffeic acid OMT fromRosa chinensis. POMT-2 was expressed inEscherichia coli as a glutathione S-transferase fusion protein, and was purified by affinity chromatography. POMT-2 transferred a methyl group
of SAM to caffeic acid and 6,7-dihydroxyflavone, but showed low activities toward quercetin and kaempferol. According to itsin vitro substrate preference and composition of phenolic compounds in poplar, thein vivo function of POMT-2 is probably the methylation of caffeic acid and an involvement in lignin biosynthesis. 相似文献
17.
Camptotheca acuminata 10‐hydroxycamptothecin O‐methyltransferase: an alkaloid biosynthetic enzyme co‐opted from flavonoid metabolism
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Vonny Salim A. Daniel Jones Dean DellaPenna 《The Plant journal : for cell and molecular biology》2018,95(1):112-125
The medicinal plant Camptotheca acuminata accumulates camptothecin, 10‐hydroxycamptothecin, and 10‐methoxycamptothecin as its major bioactive monoterpene indole alkaloids. Here, we describe identification and functional characterization of 10‐hydroxycamptothecin O‐methyltransferase (Ca10OMT), a member of the Diverse subclade of class II OMTs. Ca10OMT is highly active toward both its alkaloid substrate and a wide range of flavonoids in vitro and in this way contrasts with other alkaloid OMTs in the subclade that only utilize alkaloid substrates. Ca10OMT shows a strong preference for the A‐ring 7‐OH of flavonoids, which is structurally equivalent to the 10‐OH of 10‐hydroxycamptothecin. The substrates of other alkaloid OMTs in the subclade bear little similarity to flavonoids, but the 3‐D positioning of the 7‐OH, A‐ and C‐rings of flavonoids is nearly identical to the 10‐OH, A‐ and B‐rings of 10‐hydroxycamptothecin. This structural similarity likely explains the retention of flavonoid OMT activity by Ca10OMT and also why kaempferol and quercetin aglycones are potent inhibitors of its 10‐hydroxycamptothecin activity. The catalytic promiscuity and strong inhibition of Ca10OMT by flavonoid aglycones in vitro prompted us to investigate the potential physiological roles of the enzyme in vivo. Based on its regioselectivity, kinetic parameters and absence of 7‐OMT flavonoids in vivo, we conclude that the major and likely only substrate of Ca10OMTin vivo is 10‐hydroxycamptothecin. This is likely accomplished by Ca10OMT being kept spatially separated at the tissue levels from potentially inhibitory flavonoid aglycones, and flavonoid aglycones being rapidly glycosylated to non‐inhibitory flavonoid glycosides. 相似文献
18.
A single residue of the NAD(H)-dependent lactate dehydrogenase (LDH) from Bacillus stearothermophilus has been changed in order to decrease substrate inhibition. The conserved aspartic acid residue at position 52 was replaced by glutamate using site-directed mutagenesis. The effect on substrate inhibition was measured. In the glutamate-52 mutant substrate inhibition is decreased twofold. 相似文献
19.
Mohammed N.A. Khalil Wolfgang Brandt Till Beuerle Dennis Reckwell Josephine Groeneveld Robert Hänsch Mariam M. Gaid Benye Liu Ludger Beerhues 《The Plant journal : for cell and molecular biology》2015,83(2):263-276
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. 相似文献
20.
Sesquiterpene cyclases catalyze the conversion of common precursor, farnesyl pyrophosphate, into various terpene backbones. X-ray crystallography of tobacco epi-aristolochene synthase has previously proposed a cyclization mechanism wherein the allylic carbocation intermediate is stabilized by the main chain carbonyl oxygens of three consecutive threonine residues. Alignment of amino acid sequences of plant terpene cyclases shows that the first position of the triad is almost invariably threonine or serine. To probe the carbocation-stabilizing role, the amino acid residues of the 433TSA435 triad in (+)-germacrene A synthase from Ixeris dentata were altered by site-directed mutagenesis. Enzyme kinetic measurements of the mutants and GC/MS analysis of the enzyme reaction products indicate that mutations of the triad decreased enzyme catalysis rather than substrate binding but did not affect its structural rearrangement in the catalytic mechanism. This is the first report that the hydroxyl group of threonine at the first position of the triad is required for the cyclase activity. 相似文献