Sequence analysis and specificity of distinct types of menaquinone methyltransferases indicate the widespread potential of methylmenaquinone production in bacteria and archaea

Menaquinone (MK) serves as an essential membranous redox mediator in various electron transport chains of aerobic and anaerobic respiration. In addition, the composition of the quinone/quinol pool has been widely used as a biomarker in microbial taxonomy. The HemN-like class C radical SAM methyltransferases (RSMTs) MqnK, MenK and MenK2 have recently been shown to facilitate specific menaquinone methylation reactions at position C-8 (MqnK/MenK) or C-7 (MenK2) to synthesize 8-methylmenaquinone, 7-methylmenaquinone and 7,8-dimethylmenaquinone. However, the vast majority of protein sequences from the MqnK/MenK/MenK2 family belong to organisms, whose capacity to produce methylated menaquinones has not been investigated biochemically. Here, representative putative menK and menK2 genes from Collinsella tanakaei and Ferrimonas marina were individually expressed in Escherichia coli (wild-type or ubiE deletion mutant) and the corresponding cells were found to produce methylated derivatives of the endogenous MK and 2-demethylmenaquinone. Cluster and phylogenetic analyses of 828 (methyl)menaquinone methyltransferase sequences revealed signature motifs that allowed to discriminate enzymes of the MqnK/MenK/MenK2 family from other radical SAM enzymes and to identify C-7-specific menaquinone methyltransferases of the MenK2 subfamily. This study will help to predict the methylation status of the quinone/quinol pool of a microbial species (or even a microbial community) from its (meta)genome and contribute to the future design of microbial quinone/quinol pools in a Synthetic Biology approach.     

A class C radical S‐adenosylmethionine methyltransferase synthesizes 8‐methylmenaquinone

The membranous quinone/quinol pool is essential to the majority of life forms and has been widely used as an important biomarker in microbial taxonomy. In the anaerobic world, the most important quinones are menaquinone (MK) and a methylated form of MK, designated methylmenaquinone (MMK), which is anticipated to serve specifically in low‐potential electron transport chains involved in anaerobic respiration. However, it has remained unclear how MMK is generated. Here, we show that a novel enzyme homologous to class C radical SAM methyltransferases (RSMTs) synthesizes MMK using MK as substrate. Such enzymes, termed either MenK or MqnK, are present in MMK‐producing bacteria (and some archaea) that possess either the classical MK biosynthesis pathway (Men) or the futalosine pathway (Mqn). An mqnK deletion mutant of the model Epsilonproteobacterium Wolinella succinogenes was unable to produce MMK₆ but its formation was restored upon genomic complementation using either the native mqnK gene or menK from the human gut bacterium Adlercreutzia equolifaciens or Shewanella oneidensis. Moreover, any of the menK genes enabled Escherichia coli cells to produce MMK₈ and a methylated form of 2‐demethylmenaquinone₈ (DMK₈). The results expand the knowledge on quinone synthesis and demonstrate an unprecedented function for a class C RSMT‐type enzyme in primary cell metabolism.     

Identification of a Unique Radical S-Adenosylmethionine Methylase Likely Involved in Methanopterin Biosynthesis in Methanocaldococcus jannaschii

Methanopterin (MPT) and its analogs are coenzymes required for methanogenesis and methylotrophy in specialized microorganisms. The methyl groups at C-7 and C-9 of the pterin ring distinguish MPT from all other pterin-containing natural products. However, the enzyme(s) responsible for the addition of these methyl groups has yet to be identified. Here we demonstrate that a putative radical S-adenosyl-l-methionine (SAM) enzyme superfamily member encoded by the MJ0619 gene in the methanogen Methanocaldococcus jannaschii is likely this missing methylase. When MJ0619 was heterologously expressed in Escherichia coli, various methylated pterins were detected, consistent with MJ0619 catalyzing methylation at C-7 and C-9 of 7,8-dihydro-6-hydroxymethylpterin, a common intermediate in both folate and MPT biosynthesis. Site-directed mutagenesis of Cys77 present in the first of two canonical radical SAM CX₃CX₂C motifs present in MJ0619 did not inhibit C-7 methylation, while mutation of Cys102, found in the other radical SAM amino acid motif, resulted in the loss of C-7 methylation, suggesting that the first motif could be involved in C-9 methylation, while the second motif is required for C-7 methylation. Further experiments demonstrated that the C-7 methyl group is not derived from methionine and that methylation does not require cobalamin. When E. coli cells expressing MJ0619 were grown with deuterium-labeled acetate as the sole carbon source, the resulting methyl group on the pterin was predominantly labeled with three deuteriums. Based on these results, we propose that this archaeal radical SAM methylase employs a previously uncharacterized mechanism for methylation, using methylenetetrahydrofolate as a methyl group donor.     

Characterization of a methyltransferase involved in herboxidiene biosynthesis

The herboxidiene biosynthetic gene cluster contains a regulatory gene and six biosynthetic genes that encode three polyketide synthases (HerB, HerC and HerD) and three tailoring enzymes (HerE, HerF and HerG). Through single crossover recombination, an integrative plasmid was inserted into the genome of Streptomyces chromofuscus ATCC 49982 between herE and herF, resulting in low-level expression of herF and the downstream herG. The mutant strain produced two new compounds, 18-deoxy-25-demethyl-herboxidiene and 25-demethyl-herboxidiene. HerF was expressed in Escherichia coli and biochemically characterized as the dedicated methyltransferase in herboxidiene biosynthesis. It prefers 25-demethyl-herboxidiene to 18-deoxy-25-demethyl-herboxidiene, suggesting that C-25 methylation is the last tailoring step.     

The Corynebacterium glutamicum cglIM gene encoding a 5-cytosine methyltransferase enzyme confers a specific DNA methylation pattern in an McrBC-deficient Escherichia coli strain

The cglIM gene of the coryneform soil bacterium Corynebacterium glutamicum ATCC 13032 has been cloned and characterized. The coding region comprises 1092 nucleotides and specifies a protein of 363 amino acid residues with a deduced M_r of 40 700. The amino acid sequence showed striking similarities to methyltransferase enzymes generating 5-methylcytosine residues, especially to M·NgoVII from Neisseria gonorrhoeae recognizing the sequence GCSGC. The cglIM gene is organized in an unusual operon which contains, in addition, two genes encoding stress-sensitive restriction enzymes. Using PCR techniques the entire gene including the promoter region was amplified from the wild-type chromosome and cloned in Escherichia coli. Expression of the cglIM gene in E. coli under the control of its own promoter conferred the C. glutamicum-specific methylation pattern to co-resident shuttle plasmids and led to a 260-fold increase in the transformation rate of C. glutamicum. In addition, the methylation pattern produced by this methyltransferase enzyme is responsible for the sensitivity of DNA from C. glutamicum to the modified cytosine restriction (Mcr) system of E. coli.     

Synthesis and dopaminergic activity of a series of new 1-aryl tetrahydroisoquinolines and 2-substituted 1-aryl-3-tetrahydrobenzazepines

A series of new 1-aryl-6,7-dihydroxy tetrahydroisoquinolines with several substitution patterns in the 1-aryl group at C-1 were prepared in good yields. The influence of each substituent on the affinity and selectivity for D₁ and D₂ dopaminergic receptors was studied. Moreover, N-alkyl salts of these tetrahydroisoquinolines were used as starting material to synthesize a series of new 1-aryl-7,8-dihydroxy 3-tetrahydrobenzazepines derivatives with electron-withdrawing substituents at C-2 position by the diastereoselective Stevens rearrangement. The structure-activity relationship of these compounds was explored to evaluate the effect of the functional group at C-2 in benzazepines and the modification in the aryl group at the isoquinoline C-1 position towards the affinity and selectivity for the mentioned receptors. The 1-aryl-6,7-dihydroxy tetrahydroisoquinoline 4c shows significant affinity towards D₂ receptor, with K_i value of 31 nM. This significant affinity can be attributed to the presence of a thiomethyl group, and it is the most active 1-aryl-6,7-dihydroxy tetrahydroisoquinoline derivative reported to date.     

Competitive Lrp and Dam assembly at the pap regulatory region: implications for mechanisms of epigenetic regulation

Escherichia coli DNA adenine methyltransferase (Dam) and Leucine-responsive regulatory protein (Lrp) are key regulators of the pap operon, which codes for the pilus proteins necessary for uropathogenic E. coli cellular adhesion. The pap operon is regulated by a phase variation mechanism in which the methylation states of two GATC sites in the pap regulatory region and the binding position of Lrp determine whether the pilus genes are expressed. The post-replicative reassembly of Dam, Lrp, and the local regulator PapI onto a hemimethylated pap intermediate is a critical step of the phase variation switching mechanism and is not well understood. We show that Lrp, in the presence and in the absence of PapI and nonspecific DNA, specifically protects pap regulatory GATC sites from Dam methylation when allowed to compete with Dam for assembly on unmethylated and hemimethylated pap DNA. The methylation protection is dependent upon the concentration of Lrp and does not occur with non-regulatory GATC sites. Our data suggest that only at low Lrp concentrations will Dam compete effectively for binding and methylation of the proximal GATC site, leading to a phase switch resulting in the expression of pili.     

Production of a Doubly Chiral Compound, (4R,6R)-4-Hydroxy-2,2,6-Trimethylcyclohexanone, by Two-Step Enzymatic Asymmetric Reduction

A practical enzymatic synthesis of a doubly chiral key compound, (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone, starting from the readily available 2,6,6-trimethyl-2-cyclohexen-1,4-dione is described. Chirality is first introduced at the C-6 position by a stereoselective enzymatic hydrogenation of the double bond using old yellow enzyme 2 of Saccharomyces cerevisiae, expressed in Escherichia coli, as a biocatalyst. Thereafter, the carbonyl group at the C-4 position is reduced selectively and stereospecifically by levodione reductase of Corynebacterium aquaticum M-13, expressed in E. coli, to the corresponding alcohol. Commercially available glucose dehydrogenase was also used for cofactor regeneration in both steps. Using this two-step enzymatic asymmetric reduction system, 9.5 mg of (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone/ml was produced almost stoichiometrically, with 94% enantiomeric excess in the presence of glucose, NAD⁺, and glucose dehydrogenase. To our knowledge, this is the first report of the application of S. cerevisiae old yellow enzyme for the production of a useful compound.     

Effects of pyrogallol on O-methylation of dopamine and salsolinol

In the cultured cells of Corydalis pallida var. tenuis, the formation of 6-O-methylated metabolites (6-D₄ and 6-D₇) from salsolinol-D₄ (2-D₄) was reduced by pyrogallol. The production of 3-O-methylated derivatives (10 and 10-D₃) from dopamine was almost not affected by pyrogallol. Similar results were obtained in intact plants, though the effect of pyrogallol on methylation of C-6-OH of salsolinol in plants is smaller than that in the cultured cells. These results show that the effects of pyrogallol on the methylation of C-6-OH of salsolinol and that on the methylation of C-3-OH of dopamine are different, suggesting that the O-methylating enzymes of salsolinol and dopamine are different in C. pallida var. tenuis. The production of 3-O-methyldopamine in the presence of pyrogallol was reduced in intact plants of Cynanchum vincetoxicum, but not in the cultured cells. The effect of pyrogallol on the methylation of salsolinol was uncertain in Cyn. vincetoxicum. Both 6- and 7-O-methylations of salsolinol occur in Cyn. vincetoxicum, while only 6-O-methylation occurs in C. pallida var. tenuis. This could reveal that the O-methylating enzymes at C-6 and C-7 are different.     

The yfiC gene of E. coli encodes an adenine-N6 methyltransferase that specifically modifies A37 of tRNA1Val(cmo5UAC)

Transfer RNA is highly modified. Nucleotide 37 of the anticodon loop is represented by various modified nucleotides. In Escherichia coli, the valine-specific tRNA (cmo⁵UAC) contains a unique modification, N⁶-methyladenosine, at position 37; however, the enzyme responsible for this modification is unknown. Here we demonstrate that the yfiC gene of E. coli encodes an enzyme responsible for the methylation of A37 in tRNA₁^Val. Inactivation of yfiC gene abolishes m⁶A formation in tRNA₁^Val, while expression of the yfiC gene from a plasmid restores the modification. Additionally, unmodified tRNA₁^Val can be methylated by recombinant YfiC protein in vitro. Although the methylation of m⁶A in tRNA₁^Val by YfiC has little influence on the cell growth under standard conditions, the yfiC gene confers a growth advantage under conditions of osmotic and oxidative stress.     

Heterologous Escherichia coli Expression, Purification and Characterization of the GrmA Aminoglycoside-Resistance Methyltransferase

The mechanism of resistance to aminoglycosides based on methylation of their target, 16S rRNA, was until recently described only in antibiotic producing microorganisms. However, equivalent methyltransferases have now also been identified among numerous clinical Gram-negative pathogenic isolates. We have cloned, expressed, and purified GrmA, the aminoglycoside-resistance methyltransferase from Micromonospora purpurea, producer of gentamicin complex. Two vectors were created that express protein with an N-terminal 6× histidine tag with and without an enterokinase recognition producing proteins His₆-EK-GrmA and His₆-GrmA, respectively. The activity of both recombinant proteins was demonstrated in vivo. After optimized expression and native purification both protein variants proved to be active in in vitro methylation assays. This work lays a foundation for future detailed biochemical, structural and pharmacological studies with this member of an important group of aminoglycoside-resistance enzymes.     

Highly stereoselective synthesis of C-vinyl furanosides through acid-catalyzed S(N)2 inversion at the C-3 position of 1,2-dideoxy-hept-1-enitols

A highly stereoselective synthesis of C-vinyl furanosides through the S_N2 inversion at the C-3 position of the 1,2-dideoxy-hept-1-enitols is disclosed. Treatment of the 1,2-dideoxy-hept-1-enitols with diphenylammonium trifluoromethanesulfonate as the acid catalyst produced the C-vinyl furanosides (3,6-anhydro-1,2-dideoxy-hept-1-enitol derivatives) via a subsequent S_N2 intramolecular debenzyloxyation-cycloetherification reaction at the C-3 position.     

A simple and rapid thin-layer chromatography method to follow the kinetics of product formation by 7,8-dihydro-6-hydroxymethylpterin pyrophosphokinase and 7,8-dihydropteroate synthetase.

A rapid, simple, and inexpensive thin-layer chromatography method was developed to follow and to quantify enzymatic reactions of the title enzymes without need of a microbiological test system. The method was used to measure the kinetics of product formation by the folate-synthesizing enzymes 7,8-dihydro-6-hydroxymethylpterin pyrophosphokinase (I) and 7,8-dihydropteroate synthetase (II) of Escherichia coli. Thus test conditions could be optimized for the reaction of H₂PtCH₂OH to H₂PtCH₂OPP by I and of H₂PtCH₂OH to H₂-pteroic acid as well as its derivatives by I plus II. Furthermore this tle system allows direct measurements of I and/or II activity in fractions obtained by column chromatography (gel filtration and ion exchange, respectively).     

YibK is the 2′-O-methyltransferase TrmL that modifies the wobble nucleotide in Escherichia coli tRNALeu isoacceptors

Transfer RNAs are the most densely modified nucleic acid molecules in living cells. In Escherichia coli, more than 30 nucleoside modifications have been characterized, ranging from methylations and pseudouridylations to more complex additions that require multiple enzymatic steps. Most of the modifying enzymes have been identified, although a few notable exceptions include the 2′-O-methyltransferase(s) that methylate the ribose at the nucleotide 34 wobble position in the two leucyl isoacceptors tRNA^Leu_CmAA and tRNA^Leu_cmnm5UmAA. Here, we have used a comparative genomics approach to uncover candidate E. coli genes for the missing enzyme(s). Transfer RNAs from null mutants for candidate genes were analyzed by mass spectrometry and revealed that inactivation of yibK leads to loss of 2′-O-methylation at position 34 in both tRNA^Leu_CmAA and tRNA^Leu_cmnm5UmAA. Loss of YibK methylation reduces the efficiency of codon–wobble base interaction, as demonstrated in an amber suppressor supP system. Inactivation of yibK had no detectable effect on steady-state growth rate, although a distinct disadvantage was noted in multiple-round, mixed-population growth experiments, suggesting that the ability to recover from the stationary phase was impaired. Methylation is restored in vivo by complementing with a recombinant copy of yibK. Despite being one of the smallest characterized α/β knot proteins, YibK independently catalyzes the methyl transfer from S-adenosyl-L-methionine to the 2′-OH of the wobble nucleotide; YibK recognition of this target requires a pyridine at position 34 and N⁶-(isopentenyl)-2-methylthioadenosine at position 37. YibK is one of the last remaining E. coli tRNA modification enzymes to be identified and is now renamed TrmL.     

KsgA, a 16S rRNA adenine methyltransferase, has a novel DNA glycosylase/AP lyase activity to prevent mutations in Escherichia coli

The 5-formyluracil (5-foU), a major mutagenic oxidative damage of thymine, is removed from DNA by Nth, Nei and MutM in Escherichia coli. However, DNA polymerases can also replicate past the 5-foU by incorporating C and G opposite the lesion, although the mechanism of correction of the incorporated bases is still unknown. In this study, using a borohydride-trapping assay, we identified a protein trapped by a 5-foU/C-containing oligonucleotide in an extract from E. coli mutM nth nei mutant. The protein was subsequently purified from the E. coli mutM nth nei mutant and was identified as KsgA, a 16S rRNA adenine methyltransferase. Recombinant KsgA also formed the trapped complex with 5-foU/C- and thymine glycol (Tg)/C-containing oligonucleotides. Furthermore, KsgA excised C opposite 5-foU, Tg and 5-hydroxymethyluracil (5-hmU) from duplex oligonucleotides via a β-elimination reaction, whereas it could not remove the damaged base. In contrast, KsgA did not remove C opposite normal bases, 7,8-dihydro-8-oxoguanine and 2-hydroxyadenine. Finally, the introduction of the ksgA mutation increased spontaneous mutations in E. coli mutM mutY and nth nei mutants. These results demonstrate that KsgA has a novel DNA glycosylase/AP lyase activity for C mispaired with oxidized T that prevents the formation of mutations, which is in addition to its known rRNA adenine methyltransferase activity essential for ribosome biogenesis.     

YbeA is the m3Psi methyltransferase RlmH that targets nucleotide 1915 in 23S rRNA

Pseudouridines in the stable RNAs of Bacteria are seldom subjected to further modification. There are 11 pseudouridine (Ψ) sites in Escherichia coli rRNA, and further modification is found only at Ψ1915 in 23S rRNA, where the N-3 position of the base becomes methylated. Here, we report the identity of the E. coli methyltransferase that specifically catalyzes methyl group addition to form m³Ψ1915. Analyses of E. coli rRNAs using MALDI mass spectrometry showed that inactivation of the ybeA gene leads to loss of methylation at nucleotide Ψ1915. Methylation is restored by complementing the knockout strain with a plasmid-encoded copy of ybeA. Homologs of the ybeA gene, and thus presumably the ensuing methylation at nucleotide m³Ψ1915, are present in most bacterial lineages but are essentially absent in the Archaea and Eukaryota. Loss of ybeA function in E. coli causes a slight slowing of the growth rate. Phylogenetically, ybeA and its homologs are grouped with other putative S-adenosylmethionine-dependent, SPOUT methyltransferase genes in the Cluster of Orthologous Genes COG1576; ybeA is the first member to be functionally characterized. The YbeA methyltransferase is active as a homodimer and docks comfortably into the ribosomal A site without encroaching into the P site. YbeA makes extensive interface contacts with both the 30S and 50S subunits to align its active site cofactor adjacent to nucleotide Ψ1915. Methylation by YbeA (redesignated RlmH for rRNA large subunit methyltransferase H) possibly functions as a stamp of approval signifying that the 50S subunit has engaged in translational initiation.     

Polyunsaturated C18 fatty acids derivatized with Gly and Ile as an additional tool for studies of the catalytic evolution of fungal 8- and 9-dioxygenases

The fungal linoleate diol synthase (LDS) family contains over twenty characterized 8-, 9-, and 10-dioxygenases (DOX), usually fused to catalytically competent cytochromes P450. Crystal structures are not available, but indirect evidence suggests that linoleic acid enters the active site of 8R-DOX-LDS headfirst and enters 9S-DOX-allene oxide synthase (AOS) with the ω-end (tail) first. Fatty acids derivatized with amino acids can conceivably be used to study oxidation in tail first position by enzymes, which bind natural fatty acids headfirst. The results might reveal catalytic similarities of homologous enzymes. 8R-DOX-5,8-LDS oxidize 18:2n-6-Ile and 18:2n-6-Gly in tail first position to 9S-hydroperoxy metabolites, albeit with less position and stereo specificity than 9S-DOX-AOS. The oxygenation mechanism of 9S-DOX-AOS with antarafacial hydrogen abstraction at C-11 and oxygen insertion at C-9 was also retained. Two homologues, 8R-DOX-7,8-LDS and 8R-DOX-AOS, oxidized 18:2n-6-Ile and 18:2n-6-Gly at C-9, suggesting a conserved feature of 8R-DOX domains. 9R-DOX-AOS, with 54% sequence identity to 9S-DOX-AOS, did not oxidize the derivatized C₁₈ fatty acids. 9Z,12Z-16:2, two carbon shorter than 18:n-6 from the ω-end, was rapidly metabolized to an α-ketol, but 7Z,10Z-16:2 was not a substrate. An unsaturated carbon chain from C-1 to C-8 was apparently more important than the configuration at the ω-end. 8R-DOX-LDS and 9R-DOX-AOS may thus bind 18:2n-6 in the same orientation. The oxidation of 18:2n-6 in straight or reverse head-to-tail positions illustrates evolutionary traits between 8- and 9-DOX domains. Fatty acids derivatized with amino acids provide a complementary tool for the analysis of evolution of enzymes.     

Characterization of S-Adenosylmethionine: Ribosomal Ribonucleic Acid-Adenine (N6-) Methyltransferase of Escherichia coli Strain B

This study is concerned with the isolation and characterization of the enzyme, S-adenosylmethionine:ribosomal ribonucleic acid-adenine (N⁶⁻) methyl-transferase [rRNA-adenine (N⁶-) methylase] of Escherichia coli strain B, which is responsible for the formation of N⁶-methyladenine moieties in ribosomal ribonucleic acids (rRNA). A 1,500-fold purified preparation of the species-specific methyltransferase methylates a limited number of adenine moieties in heterologous rRNA (Micrococcus lysodeikticus and Bacillus subtilis) and methyl-deficient homologous rRNA. The site recognition mechanism does not require intact 16 or 23S rRNA. The enzyme does not utilize transfer ribonucleic acid as a methyl acceptor nor does it synthesize 2-methyladenine or N⁶-dimethyladenine moieties. Mg²⁺, spermine, K⁺, and Na⁺ increase the reaction rate but not the extent of methylation; elevated concentrations of the cations inhibit markedly. The purified preparations utilize 9-β-ribosyl-2,6-diaminopurine (DAPR) as a methyl acceptor with the synthesis of 9-β-ribosyl-6-amino-2-methylaminopurine. A comparison of the two activities demonstrated that one methyltransferase is responsible for the methylation of both DAPR and rRNA. This property provides a sensitive assay procedure unaffected by ribonucleases and independent of any specificity exhibited by rRNA methyl acceptors.