Antibiotic resistance evolved via inactivation of a ribosomal RNA methylating enzyme

Modifications of the bacterial ribosome regulate the function of the ribosome and modulate its susceptibility to antibiotics. By modifying a highly conserved adenosine A2503 in 23S rRNA, methylating enzyme Cfr confers resistance to a range of ribosome-targeting antibiotics. The same adenosine is also methylated by RlmN, an enzyme widely distributed among bacteria. While RlmN modifies C2, Cfr modifies the C8 position of A2503. Shared nucleotide substrate and phylogenetic relationship between RlmN and Cfr prompted us to investigate evolutionary origin of antibiotic resistance in this enzyme family. Using directed evolution of RlmN under antibiotic selection, we obtained RlmN variants that mediate low-level resistance. Surprisingly, these variants confer resistance not through the Cfr-like C8 methylation, but via inhibition of the endogenous RlmN C2 methylation of A2503. Detection of RlmN inactivating mutations in clinical resistance isolates suggests that the mechanism used by the in vitro evolved variants is also relevant in a clinical setting. Additionally, as indicated by a phylogenetic analysis, it appears that Cfr did not diverge from the RlmN family but from another distinct family of predicted radical SAM methylating enzymes whose function remains unknown.     

Discovery and Characterization of BlsE,a Radical S-Adenosyl-L-methionine Decarboxylase Involved in the Blasticidin S Biosynthetic Pathway

BlsE, a predicted radical S-adenosyl-L-methionine (SAM) protein, was anaerobically purified and reconstituted in vitro to study its function in the blasticidin S biosynthetic pathway. The putative role of BlsE was elucidated based on bioinformatics analysis, genetic inactivation and biochemical characterization. Biochemical results showed that BlsE is a SAM-dependent radical enzyme that utilizes cytosylglucuronic acid, the accumulated intermediate metabolite in blsE mutant, as substrate and catalyzes decarboxylation at the C5 position of the glucoside residue to yield cytosylarabinopyranose. Additionally, we report the purification and reconstitution of BlsE, characterization of its [4Fe–4S] cluster using UV-vis and electron paramagnetic resonance (EPR) spectroscopic analysis, and investigation of the ability of flavodoxin (Fld), flavodoxin reductase (Fpr) and NADPH to reduce the [4Fe–4S]²⁺ cluster. Mutagenesis studies demonstrated that Cys₃₁, Cys_35, Cys₃₈ in the C×××C×MC motif and Gly₇₃, Gly₇₄, Glu₇₅, Pro₇₆ in the GGEP motif were crucial amino acids for BlsE activity while mutation of Met₃₇ had little effect on its function. Our results indicate that BlsE represents a typical [4Fe–4S]-containing radical SAM enzyme and it catalyzes decarboxylation in blasticidin S biosynthesis.     

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.     

Shared requirements for key residues in the antibiotic resistance enzymes ErmC and ErmE suggest a common mode of RNA recognition

Erythromycin-resistance methyltransferases are SAM dependent Rossmann fold methyltransferases that convert A2058 of 23S rRNA to m⁶ ₂A2058. This modification sterically blocks binding of several classes of antibiotics to 23S rRNA, resulting in a multidrug-resistant phenotype in bacteria expressing the enzyme. ErmC is an erythromycin resistance methyltransferase found in many Gram-positive pathogens, whereas ErmE is found in the soil bacterium that biosynthesizes erythromycin. Whether ErmC and ErmE, which possess only 24% sequence identity, use similar structural elements for rRNA substrate recognition and positioning is not known. To investigate this question, we used structural data from related proteins to guide site-saturation mutagenesis of key residues and characterized selected variants by antibiotic susceptibility testing, single turnover kinetics, and RNA affinity–binding assays. We demonstrate that residues in α4, α5, and the α5-α6 linker are essential for methyltransferase function, including an aromatic residue on α4 that likely forms stacking interactions with the substrate adenosine and basic residues in α5 and the α5-α6 linker that likely mediate conformational rearrangements in the protein and cognate rRNA upon interaction. The functional studies led us to a new structural model for the ErmC or ErmE-rRNA complex.     

A QM/MM study of the catalytic mechanism of SAM methyltransferase RlmN from Escherichia coli

RlmN is a radical S‐adenosylmethionine (SAM) enzyme that catalyzes the C2 methylation of adenosine 2503 (A2503) in 23S rRNA and adenosine 37 (A37) in several Escherichia coli transfer RNAs (tRNA). The catalytic reaction of RlmN is distinctly different from that of typical SAM‐dependent methyltransferases that employs an S_N2 mechanism, but follows a ping‐pong mechanism which involves the intermediate methylation of a conserved cysteine residue. Recently, the x‐ray structure of a key intermediate in the RlmN reaction has been reported, allowing us to perform combined quantum mechanics and molecular mechanics (QM/MM) calculations to delineate the reaction details of RlmN at atomic level. Starting from the Cross‐Linked RlmN C118A?tRNA complex, the possible mechanisms for both the formation and the resolution of the cross‐linked species (IM2) have been illuminated. On the basis of our calculations, IM2 is formed by the attack of the C355‐based methylene radical on the sp²‐hybridized C2 of the adenosine ring, corresponding to energy barrier of 14.4 kcal/mol, and the resolution of IM2 is confirmed to follow a radical fragmentation mechanism. The cleavage of C′–S′ bond of mC355‐A37 cross‐link is in concert with the deprotonation of C2 by C118 residue, which is the rate‐limiting step with an energy barrier of 17.4 kcal/mol. Moreover, the cleavage of C′–S′ bond of IM2 can occur independently, that is, it does not require the loss of an electron of IM2 and the formation of disulfide bond between C355 and C118 as precondition. These findings would deepen the understanding of the catalysis of RlmN.     

Cytoskeletal confinement of CX3CL1 limits its susceptibility to proteolytic cleavage by ADAM10

CX₃CL1 is a unique chemokine that acts both as a transmembrane endothelial adhesion molecule and, upon proteolytic cleavage, a soluble chemoattractant for circulating leukocytes. The constitutive release of soluble CX₃CL1 requires the interaction of its transmembrane species with the integral membrane metalloprotease ADAM10, yet the mechanisms governing this process remain elusive. Using single-particle tracking and subdiffraction imaging, we studied how ADAM10 interacts with CX₃CL1. We observed that the majority of cell surface CX₃CL1 diffused within restricted confinement regions structured by the cortical actin cytoskeleton. These confinement regions sequestered CX₃CL1 from ADAM10, precluding their association. Disruption of the actin cytoskeleton reduced CX₃CL1 confinement and increased CX₃CL1–ADAM10 interactions, promoting the release of soluble chemokine. Our results demonstrate a novel role for the cytoskeleton in limiting membrane protein proteolysis, thereby regulating both cell surface levels and the release of soluble ligand.     

Crystal structure of the N‐terminal methyltransferase‐like domain of anamorsin

Anamorsin is a recently identified molecule that inhibits apoptosis during hematopoiesis. It contains an N‐terminal methyltransferase‐like domain and a C‐terminal Fe‐S cluster motif. Not much is known about the function of the protein. To better understand the function of anamorsin, we have solved the crystal structure of the N‐terminal domain at 1.8 Å resolution. Although the overall structure resembles a typical S‐adenosylmethionine (SAM) dependent methyltransferase fold, it lacks one α‐helix and one β‐strand. As a result, the N‐terminal domain as well as the full‐length anamorsin did not show S‐adenosyl‐l ‐methionine (AdoMet) dependent methyltransferase activity. Structural comparisons with known AdoMet dependent methyltransferases reveals subtle differences in the SAM binding pocket that preclude the N‐terminal domain from binding to AdoMet. The N‐terminal methyltransferase‐like domain of anamorsin probably functions as a structural scaffold to inhibit methyl transfers by out‐competing other AdoMet dependant methyltransferases or acts as bait for protein–protein interactions.Proteins 2014; 82:1066–1071. © 2013 Wiley Periodicals, Inc.     

Rhodobacter capsulatus magnesium chelatase subunit BchH contains an oxygen sensitive iron–sulfur cluster

Magnesium chelatase is the first unique enzyme of the bacteriochlorophyll biosynthetic pathway. It consists of three subunits (BchI, BchD, and BchH). Amino acid sequence analysis of the Rhodobacter capsulatus BchH revealed a novel cysteine motif (³⁹³CX₂CX₃CX₁₄C) that was found in only six other proteobacteria (CX₂CX₃CX_11–14C). The cysteine motif is likely to coordinate an unprecedented [Fe–S] cluster. Purified BchH demonstrated absorbance in the 460 nm region. This absorbance was abolished in BchH proteins with alanine substitutions at positions Cys396 and Cys414. These modified proteins were also EPR silent. In contrast, wild type BchH protein in the reduced state showed EPR signals resembling those of a [4Fe–4S] cluster with rhombic symmetry and g values at 1.90, 1.93, and 2.09, superimposed with a [3Fe–4S] cluster centered at g = 2.02. The [3Fe–4S] signal was observed independently of the [4Fe–4S] signal under oxidizing conditions. Mg-chelatase activity assays showed that the cluster is not catalytic. We suggest that the [4Fe–4S] and [3Fe–4S] signals originate from a single coordination site on the monomeric BchH protein and that the [4Fe–4S] cluster is sensitive to oxidation. It is speculated that the cluster participates in the switching between aerobic and anaerobic life of the proteobacteria.     

Cysteine Methylation Controls Radical Generation in the Cfr Radical AdoMet rRNA Methyltransferase

The ‘radical S-adenosyl-L-methionine (AdoMet)’ enzyme Cfr methylates adenosine 2503 of the 23S rRNA in the peptidyltransferase centre (P-site) of the bacterial ribosome. This modification protects host bacteria, notably methicillin-resistant Staphylococcus aureus (MRSA), from numerous antibiotics, including agents (e.g. linezolid, retapamulin) that were developed to treat such organisms. Cfr contains a single [4Fe-4S] cluster that binds two separate molecules of AdoMet during the reaction cycle. These are used sequentially to first methylate a cysteine residue, Cys338; and subsequently generate an oxidative radical intermediate that facilitates methyl transfer to the unreactive C8 (and/or C2) carbon centres of adenosine 2503. How the Cfr active site, with its single [4Fe-4S] cluster, catalyses these two distinct activities that each utilise AdoMet as a substrate remains to be established. Here, we use absorbance and electron paramagnetic resonance (EPR) spectroscopy to investigate the interactions of AdoMet with the [4Fe-4S] clusters of wild-type Cfr and a Cys338 Ala mutant, which is unable to accept a methyl group. Cfr binds AdoMet with high (∼ 10 µM) affinity notwithstanding the absence of the RNA cosubstrate. In wild-type Cfr, where Cys338 is methylated, AdoMet binding leads to rapid oxidation of the [4Fe-4S] cluster and production of 5''-deoxyadenosine (DOA). In contrast, while Cys338 Ala Cfr binds AdoMet with equivalent affinity, oxidation of the [4Fe-4S] cluster is not observed. Our results indicate that the presence of a methyl group on Cfr Cys338 is a key determinant of the activity of the enzyme towards AdoMet, thus enabling a single active site to support two distinct modes of AdoMet cleavage.     

The Escherichia coli RlmN methyltransferase is a dual-specificity enzyme that modifies both rRNA and tRNA and controls translational accuracy

Modifying RNA enzymes are highly specific for substrate-rRNA or tRNA-and the target position. In Escherichia coli, there are very few multisite acting enzymes, and only one rRNA/tRNA dual-specificity enzyme, pseudouridine synthase RluA, has been identified to date. Among the tRNA-modifying enzymes, the methyltransferase responsible for the m(2)A synthesis at purine 37 in a tRNA set still remains unknown. m(2)A is also present at position 2503 in the peptidyl transferase center of 23S RNA, where it is introduced by RlmN, a radical S-adenosyl-L-methionine (SAM) enzyme. Here, we show that E. coli RlmN is a dual-specificity enzyme that catalyzes methylation of both rRNA and tRNA. The ΔrlmN mutant lacks m(2)A in both RNA types, whereas the expression of recombinant RlmN from a plasmid introduced into this mutant restores tRNA modification. Moreover, RlmN performs m(2)A(37) synthesis in vitro using a tRNA chimera as a substrate. This chimera has also proved useful to characterize some tRNA identity determinants for RlmN and other tRNA modification enzymes. Our data suggest that RlmN works in a late step during tRNA maturation by recognizing a precise 3D structure of tRNA. RlmN inactivation increases the misreading of a UAG stop codon. Since loss of m(2)A(37) from tRNA is expected to produce a hyperaccurate phenotype, we believe that the error-prone phenotype exhibited by the ΔrlmN mutant is due to loss of m(2)A from 23S rRNA and, accordingly, that the m(2)A2503 modification plays a crucial role in the proofreading step occurring at the peptidyl transferase center.     

Functional dichotomy in the 16S rRNA (m1A1408) methyltransferase family and control of catalytic activity via a novel tryptophan mediated loop reorganization

Methylation of the bacterial small ribosomal subunit (16S) rRNA on the N1 position of A1408 confers exceptionally high-level resistance to a broad spectrum of aminoglycoside antibiotics. Here, we present a detailed structural and functional analysis of the Catenulisporales acidiphilia 16S rRNA (m¹A1408) methyltransferase (‘CacKam’). The apo CacKam structure closely resembles other m¹A1408 methyltransferases within its conserved SAM-binding fold but the region linking core β strands 6 and 7 (the ‘β6/7 linker’) has a unique, extended structure that partially occludes the putative 16S rRNA binding surface, and sequesters the conserved and functionally critical W203 outside of the CacKam active site. Substitution of conserved residues in the SAM binding pocket reveals a functional dichotomy in the 16S rRNA (m¹A1408) methyltransferase family, with two apparently distinct molecular mechanisms coupling cosubstrate/ substrate binding to catalytic activity. Our results additionally suggest that CacKam exploits the W203-mediated remodeling of the β6/7 linker as a novel mechanism to control 30S substrate recognition and enzymatic turnover.     

Dre2, a conserved eukaryotic Fe/S cluster protein, functions in cytosolic Fe/S protein biogenesis

In a forward genetic screen for interaction with mitochondrial iron carrier proteins in Saccharomyces cerevisiae, a hypomorphic mutation of the essential DRE2 gene was found to confer lethality when combined with Δmrs3 and Δmrs4. The dre2 mutant or Dre2-depleted cells were deficient in cytosolic Fe/S cluster protein activities while maintaining mitochondrial Fe/S clusters. The Dre2 amino acid sequence was evolutionarily conserved, and cysteine motifs (CX₂CXC and twin CX₂C) in human and yeast proteins were perfectly aligned. The human Dre2 homolog (implicated in blocking apoptosis and called CIAPIN1 or anamorsin) was able to complement the nonviability of a Δdre2 deletion strain. The Dre2 protein with triple hemagglutinin tag was located in the cytoplasm and in the mitochondrial intermembrane space. Yeast Dre2 overexpressed and purified from bacteria was brown and exhibited signature absorption and electron paramagnetic resonance spectra, indicating the presence of both [2Fe-2S] and [4Fe-4S] clusters. Thus, Dre2 is an essential conserved Fe/S cluster protein implicated in extramitochondrial Fe/S cluster assembly, similar to other components of the so-called CIA (cytoplasmic Fe/S cluster assembly) pathway although partially localized to the mitochondrial intermembrane space.     

Structural insights into the function of aminoglycoside-resistance A1408 16S rRNA methyltransferases from antibiotic-producing and human pathogenic bacteria

X-ray crystal structures were determined of the broad-spectrum aminoglycoside-resistance A1408 16S rRNA methyltransferases KamB and NpmA, from the aminoglycoside-producer Streptoalloteichus tenebrarius and human pathogenic Escherichia coli, respectively. Consistent with their common function, both are Class I methyltransferases with additional highly conserved structural motifs that embellish the core SAM-binding fold. In overall structure, the A1408 rRNA methyltransferase were found to be most similar to a second family of Class I methyltransferases of distinct substrate specificity (m⁷G46 tRNA). Critical residues for A1408 rRNA methyltransferase activity were experimentally defined using protein mutagenesis and bacterial growth assays with kanamycin. Essential residues for SAM coenzyme binding and an extended protein surface that likely interacts with the 30S ribosomal subunit were thus revealed. The structures also suggest potential mechanisms of A1408 target nucleotide selection and positioning. We propose that a dynamic extended loop structure that is positioned adjacent to both the bound SAM and a functionally critical structural motif may mediate concerted conformational changes in rRNA and protein that underpin the specificity of target selection and activation of methyltransferase activity. These new structures provide important new insights that may provide a starting point for strategies to inhibit these emerging causes of pathogenic bacterial resistance to aminoglycosides.     

杀粉蝶菌素A1生物合成基因簇中甲基转移酶PieB2的功能

【目的】研究杀粉蝶菌素A1产生菌中甲基转移酶基因pieB2的功能。【方法】利用接合转移和同源重组双交换的方法,构建pieB2基因缺失突变株,以及利用接合转移的方法,构建回补菌株。通过高保真PCR克隆pieB2基因到表达载体pET28a上,构建质粒pJTU5997,转化入大肠杆菌E.coliBL21(DE3)/pLysE中诱导表达。利用高效液相色谱检测PieB2的体外酶活。【结果】获得了pieB2基因缺失的双交换突变株。发酵结果显示,该突变株不再产生杀粉蝶菌素A1,而是积累了一种脱甲基产物。N-末端融合组氨酸标签的PieB2在大肠杆菌中获得可溶性表达,通过体外催化证明了PieB2甲基转移酶的功能。【结论】体内遗传实验和体外生化实验证明了PieB2作为甲基转移酶在杀粉蝶菌素A1合成中的作用。     

Structural characterization of BVU_3255, a methyltransferase from human intestine antibiotic resistant pathogen Bacteroides vulgatus

Methylation is important for various cellular activities. To date, there is no report of any methyltransferase structure from the human intestine antibiotic resistant pathogen Bacteroides vulgatus. The protein BVU_3255 from B. vulgatus ATCC 8482 belongs to a SAM-dependent methyltransferase. Here, we report the crystal structure of apo BVU_3255, and its complexes with SAM and SAH, which revealed a typical class I Rossmann Fold Methyltransferase. Isothermal titration calorimetric studies showed that both SAM and SAH bind with equal affinity. The structural and sequence analysis suggested that BVU_3255 is a small molecule methyltransferase and involved in methylating the intermediates in ubiquinone biosynthesis pathway.     

Structural Determination of Functional Units of the Nucleotide Binding Domain (NBD94) of the Reticulocyte Binding Protein Py235 of Plasmodium yoelii

Background

Invasion of the red blood cells (RBC) by the merozoite of malaria parasites involves a large number of receptor ligand interactions. The reticulocyte binding protein homologue family (RH) plays an important role in erythrocyte recognition as well as virulence. Recently, it has been shown that members of RH in addition to receptor binding may also have a role as ATP/ADP sensor. A 94 kDa region named Nucleotide-Binding Domain 94 (NBD94) of Plasmodium yoelii YM, representative of the putative nucleotide binding region of RH, has been demonstrated to bind ATP and ADP selectively. Binding of ATP or ADP induced nucleotide-dependent structural changes in the C-terminal hinge-region of NBD94, and directly impacted on the RBC binding ability of RH.

Methodology/Principal Findings

In order to find the smallest structural unit, able to bind nucleotides, and its coupling module, the hinge region, three truncated domains of NBD94 have been generated, termed NBD94_444–547, NBD94_566–663 and NBD94_674–793, respectively. Using fluorescence correlation spectroscopy NBD94_444–547 has been identified to form the smallest nucleotide binding segment, sensitive for ATP and ADP, which became inhibited by 4-Chloro-7-nitrobenzofurazan. The shape of NBD94_444–547 in solution was calculated from small-angle X-ray scattering data, revealing an elongated molecule, comprised of two globular domains, connected by a spiral segment of about 73.1 Å in length. The high quality of the constructs, forming the hinge-region, NBD94_566–663 and NBD94_674–793 enabled to determine the first crystallographic and solution structure, respectively. The crystal structure of NBD94_566–663 consists of two helices with 97.8 Å and 48.6 Å in length, linked by a loop. By comparison, the low resolution structure of NBD94_674–793 in solution represents a chair–like shape with three architectural segments.

Conclusions

These structures give the first insight into how nucleotide binding impacts on the overall structure of RH and demonstrates the potential use of this region as a novel drug target.     

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.     

Identification of the rrmA Gene Encoding the 23S rRNA m1G745 Methyltransferase in Escherichia coli and Characterization of an m1G745-Deficient Mutant

An Escherichia coli mutant lacking the modified nucleotide m¹G in rRNA has previously been isolated (G. R. Björk and L. A. Isaksson, J. Mol. Biol. 51:83–100, 1970). In this study, we localize the position of the m¹G to nucleotide 745 in 23S rRNA and characterize a mutant deficient in this modification. This mutant shows a 40% decreased growth rate in rich media, a drastic reduction in loosely coupled ribosomes, a 20% decreased polypeptide chain elongation rate, and increased resistance to the ribosome binding antibiotic viomycin. The rrmA gene encoding 23S rRNA m¹G745 methyltransferase was mapped to bp 1904000 on the E. coli chromosome and identified to be identical to the previously sequenced gene yebH.