Crystal structure of ErmE - 23S rRNA methyltransferase in macrolide resistance

Abstract Pathogens often receive antibiotic resistance genes through horizontal gene transfer from bacteria that produce natural antibiotics. ErmE is a methyltransferase (MTase) from Saccharopolyspora erythraea that dimethylates A2058 in 23S rRNA using S-adenosyl methionine (SAM) as methyl donor, protecting the ribosomes from macrolide binding. To gain insights into the mechanism of macrolide resistance, the crystal structure of ErmE was determined to 1.75 Å resolution. ErmE consists of an N-terminal Rossmann-like α/ß catalytic domain and a C-terminal helical domain. Comparison with ErmC’ that despite only 24% sequence identity has the same function, reveals highly similar catalytic domains. Accordingly, superposition with the catalytic domain of ErmC’ in complex with SAM suggests that the cofactor binding site is conserved. The two structures mainly differ in the C-terminal domain, which in ErmE contains a longer loop harboring an additional 310 helix that interacts with the catalytic domain to stabilize the tertiary structure. Notably, ErmE also differs from ErmC’ by having long disordered extensions at its N- and C-termini. A C-terminal disordered region rich in arginine and glycine is also a present in two other MTases, PikR1 and PikR2, which share about 30% sequence identity with ErmE and methylate the same nucleotide in 23S rRNA.

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Structural comparisons lead to the definition of a new superfamily of NAD(P)(H)-accepting oxidoreductases: the single-domain reductases/epimerases/dehydrogenases (the ‘RED’ family)

Biochemical Journal ◽

10.1042/bj3040095 ◽

1994 ◽

Vol 304 (1) ◽

pp. 95-99 ◽

Cited By ~ 56

Author(s):

G Labesse ◽

A Vidal-Cros ◽

J Chomilier ◽

M Gaudry ◽

J P Mornon

Keyword(s):

Amino Acids ◽

Sequence Alignment ◽

Active Site ◽

Tertiary Structure ◽

Binding Specificity ◽

Single Domain ◽

Divergent Evolution ◽

Cofactor Binding ◽

Sequence Identity ◽

Definition Of

Using both primary- and tertiary-structure comparisons, we have established new structural similarities shared by reductases, epimerases and dehydrogenases not previously known to be related. Despite the low sequence identity (down to 10%), short consensus segments are identified. We show that the sequence, the active site and the supersecondary structure are well conserved in these proteins. New homologues (the protochlorophyllide reductases) are detected, and we define a new superfamily composed of single-domain dinucleotide-binding enzymes. Rules for the cofactor-binding specificity are deduced from our sequence alignment. The involvement of some amino acids in catalysis is discussed. Comparison with two-domain dehydrogenases allows us to distinguish two general mechanisms of divergent evolution.

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Domain Organization and Crystal Structure of the Catalytic Domain of E.coli RluF, a Pseudouridine Synthase that Acts on 23S rRNA

Journal of Molecular Biology ◽

10.1016/j.jmb.2006.04.019 ◽

2006 ◽

Vol 359 (4) ◽

pp. 998-1009 ◽

Cited By ~ 9

Author(s):

S. Sunita ◽

H. Zhenxing ◽

J. Swaathi ◽

Miroslaw Cygler ◽

Allan Matte ◽

...

Keyword(s):

Crystal Structure ◽

Catalytic Domain ◽

23S Rrna ◽

Domain Organization ◽

Pseudouridine Synthase

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Crystal structure of a putative short-chain dehydrogenase/reductase from Paraburkholderia xenovorans

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x21012632 ◽

2022 ◽

Vol 78 (1) ◽

Author(s):

Jaysón Davidson ◽

Kyndall Nicholas ◽

Jeremy Young ◽

Deborah G. Conrady ◽

Stephen Mayclin ◽

...

Keyword(s):

Crystal Structure ◽

Amino Acids ◽

Substrate Specificity ◽

Polychlorinated Biphenyls ◽

Atomic Structure ◽

Conformational Flexibility ◽

Cofactor Binding ◽

Sequence Identity ◽

Binding Cavity ◽

Short Chain Dehydrogenase

Paraburkholderia xenovorans degrades organic wastes, including polychlorinated biphenyls. The atomic structure of a putative dehydrogenase/reductase (SDR) from P. xenovorans (PxSDR) was determined in space group P21 at a resolution of 1.45 Å. PxSDR shares less than 37% sequence identity with any known structure and assembles as a prototypical SDR tetramer. As expected, there is some conformational flexibility and difference in the substrate-binding cavity, which explains the substrate specificity. Uniquely, the cofactor-binding cavity of PxSDR is not well conserved and differs from those of other SDRs. PxSDR has an additional seven amino acids that form an additional unique loop within the cofactor-binding cavity. Further studies are required to determine how these differences affect the enzymatic functions of the SDR.

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Molecular Basis of Intrinsic Macrolide Resistance in the Mycobacterium tuberculosis Complex

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.1.143-150.2004 ◽

2004 ◽

Vol 48 (1) ◽

pp. 143-150 ◽

Cited By ~ 96

Author(s):

Karolína Buriánková ◽

Florence Doucet-Populaire ◽

Olivier Dorson ◽

Anne Gondran ◽

Jean-Claude Ghnassia ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Macrolide Resistance ◽

Saccharopolyspora Erythraea ◽

23S Rrna ◽

Type I ◽

Intrinsic Resistance ◽

Streptomyces Ambofaciens ◽

Resistance Patterns ◽

Sequence Encoding ◽

Mycobacterial Cell Wall

ABSTRACT The intrinsic resistance of the Mycobacterium tuberculosis complex (MTC) to most antibiotics, including macrolides, is generally attributed to the low permeability of the mycobacterial cell wall. However, nontuberculous mycobacteria (NTM) are much more sensitive to macrolides than members of the MTC. A search for macrolide resistance determinants within the genome of M. tuberculosis revealed the presence of a sequence encoding a putative rRNA methyltransferase. The deduced protein is similar to Erm methyltransferases, which confer macrolide-lincosamide-streptogramin (MLS) resistance by methylation of 23S rRNA, and was named ErmMT. The corresponding gene, ermMT (erm37), is present in all members of the MTC but is absent in NTM species. Part of ermMT is deleted in some vaccine strains of Mycobacterium bovis BCG, such as the Pasteur strain, which lack the RD2 region. The Pasteur strain was susceptible to MLS antibiotics, whereas MTC species harboring the RD2 region were resistant to them. The expression of ermMT in the macrolide-sensitive Mycobacterium smegmatis and BCG Pasteur conferred MLS resistance. The resistance patterns and ribosomal affinity for erythromycin of Mycobacterium host strains expressing ermMT, srmA (monomethyltransferase from Streptomyces ambofaciens), and ermE (dimethyltransferase from Saccharopolyspora erythraea) were compared, and the ones conferred by ErmMT were similar to those conferred by SrmA, corresponding to the MLS type I phenotype. These results suggest that ermMT plays a major role in the intrinsic macrolide resistance of members of the MTC and could be the first example of a gene conferring resistance by target modification in mycobacteria.

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Faculty Opinions recommendation of Crystal structure of the human lymphoid tyrosine phosphatase catalytic domain: insights into redox regulation .

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1162316.622811 ◽

2009 ◽

Author(s):

Amy Barrios

Keyword(s):

Crystal Structure ◽

Redox Regulation ◽

Catalytic Domain ◽

Tyrosine Phosphatase

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Structural and functional insights into esterase-mediated macrolide resistance

Nature Communications ◽

10.1038/s41467-021-22016-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Michał Zieliński ◽

Jaeok Park ◽

Barry Sleno ◽

Albert M. Berghuis

Keyword(s):

Active Site ◽

Pathogenic Bacteria ◽

Catalytic Mechanism ◽

Three Dimensional ◽

Resistance Mechanisms ◽

Docking Studies ◽

Macrolide Resistance ◽

Flexible Docking ◽

Sequence Identity ◽

Substrate Spectrum

AbstractMacrolides are a class of antibiotics widely used in both medicine and agriculture. Unsurprisingly, as a consequence of their exensive usage a plethora of resistance mechanisms have been encountered in pathogenic bacteria. One of these resistance mechanisms entails the enzymatic cleavage of the macrolides’ macrolactone ring by erythromycin esterases (Eres). The most frequently identified Ere enzyme is EreA, which confers resistance to the majority of clinically used macrolides. Despite the role Eres play in macrolide resistance, research into this family enzymes has been sparse. Here, we report the first three-dimensional structures of an erythromycin esterase, EreC. EreC is an extremely close homologue of EreA, displaying more than 90% sequence identity. Two structures of this enzyme, in conjunction with in silico flexible docking studies and previously reported mutagenesis data allowed for the proposal of a detailed catalytic mechanism for the Ere family of enzymes, labeling them as metal-independent hydrolases. Also presented are substrate spectrum assays for different members of the Ere family. The results from these assays together with an examination of residue conservation for the macrolide binding site in Eres, suggests two distinct active site archetypes within the Ere enzyme family.

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The first crystal structure of the peptidase domain of the U32 peptidase family

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004715019549 ◽

2015 ◽

Vol 71 (12) ◽

pp. 2505-2512 ◽

Cited By ~ 4

Author(s):

Magdalena Schacherl ◽

Angelika A. M. Montada ◽

Elena Brunstein ◽

Ulrich Baumann

Keyword(s):

Crystal Structure ◽

Catalytic Mechanism ◽

Quaternary Structure ◽

Catalytic Domain ◽

Three Dimensional ◽

Zinc Ion ◽

Crystal Structure Analysis ◽

Dimensional Structure ◽

Sequence Motifs ◽

Conserved Sequence

The U32 family is a collection of over 2500 annotated peptidases in the MEROPS database with unknown catalytic mechanism. They mainly occur in bacteria and archaea, but a few representatives have also been identified in eukarya. Many of the U32 members have been linked to pathogenicity, such as proteins fromHelicobacterandSalmonella. The first crystal structure analysis of a U32 catalytic domain fromMethanopyrus kandleri(genemk0906) reveals a modified (βα)8TIM-barrel fold with some unique features. The connecting segment between strands β7 and β8 is extended and helix α7 is located on top of the C-terminal end of the barrel body. The protein exhibits a dimeric quaternary structure in which a zinc ion is symmetrically bound by histidine and cysteine side chains from both monomers. These residues reside in conserved sequence motifs. No typical proteolytic motifs are discernible in the three-dimensional structure, and biochemical assays failed to demonstrate proteolytic activity. A tunnel in which an acetate ion is bound is located in the C-terminal part of the β-barrel. Two hydrophobic grooves lead to a tunnel at the C-terminal end of the barrel in which an acetate ion is bound. One of the grooves binds to aStrep-Tag II of another dimer in the crystal lattice. Thus, these grooves may be binding sites for hydrophobic peptides or other ligands.

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Crystal structure of Pistol, a class of self-cleaving ribozyme

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1611191114 ◽

2017 ◽

Vol 114 (5) ◽

pp. 1021-1026 ◽

Cited By ~ 32

Author(s):

Laura A. Nguyen ◽

Jimin Wang ◽

Thomas A. Steitz

Keyword(s):

Crystal Structure ◽

Rna Structure ◽

Tertiary Structure ◽

Genomic Analysis ◽

Comparative Genomic ◽

Nonbridging Oxygen ◽

Close Proximity ◽

Domains Of Life ◽

Guanine Base ◽

A Minor

Small self-cleaving ribozymes have been discovered in all evolutionary domains of life. They can catalyze site-specific RNA cleavage, and as a result, they have relevance in gene regulation. Comparative genomic analysis has led to the discovery of a new class of small self-cleaving ribozymes named Pistol. We report the crystal structure of Pistol at 2.97-Å resolution. Our results suggest that the Pistol ribozyme self-cleavage mechanism likely uses a guanine base in the active site pocket to carry out the phosphoester transfer reaction. The guanine G40 is in close proximity to serve as the general base for activating the nucleophile by deprotonating the 2′-hydroxyl to initiate the reaction (phosphoester transfer). Furthermore, G40 can also establish hydrogen bonding interactions with the nonbridging oxygen of the scissile phosphate. The proximity of G32 to the O5′ leaving group suggests that G32 may putatively serve as the general acid. The RNA structure of Pistol also contains A-minor interactions, which seem to be important to maintain its tertiary structure and compact fold. Our findings expand the repertoire of ribozyme structures and highlight the conserved evolutionary mechanism used by ribozymes for catalysis.

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Molecular Basis of Macrolide Resistance inCampylobacterStrains Isolated from Poultry in South Korea

BioMed Research International ◽

10.1155/2018/4526576 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9 ◽

Cited By ~ 3

Author(s):

Bai Wei ◽

Min Kang

Keyword(s):

Molecular Mechanisms ◽

Macrolide Resistance ◽

23S Rrna ◽

Rrna Gene ◽

Campylobacter Coli ◽

Erythromycin Resistance ◽

23S Rrna Gene ◽

Ribosomal Protein L22 ◽

Resistant Strains ◽

Azithromycin Resistance

We investigated the molecular mechanisms underlying macrolide resistance in 38 strains ofCampylobacterisolated from poultry. Twenty-seven strains were resistant to azithromycin and erythromycin, five showed intermediate azithromycin resistance and erythromycin susceptibility, and six showed azithromycin resistance and erythromycin susceptibility. FourCampylobacter jejuniand sixCampylobacter colistrains had azithromycin MICs which were 8–16 and 2–8-fold greater than those of erythromycin, respectively. The A2075G mutation in the 23S rRNA gene was detected in 11 resistant strains with MICs ranging from 64 to ≥ 512μg/mL. Mutations including V137A, V137S, and a six-amino acid insertion (114-VAKKAP-115) in ribosomal protein L22 were detected in theC. jejunistrains. Erythromycin ribosome methylase B-erm(B) was not detected in any strain. All strains except three showed increased susceptibility to erythromycin with twofold to 256-fold MIC change in the presence of phenylalanine arginine ß-naphthylamide (PAßN); the effects of PAßN on azithromycin MICs were limited in comparison to those on erythromycin MICs, and 13 strains showed no azithromycin MIC change in the presence of PAßN. Differences between azithromycin and erythromycin resistance and macrolide resistance phenotypes and genotypes were observed even in highly resistant strains. Further studies are required to better understand macrolide resistance inCampylobacter.

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