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Antibiotic Resistance and the Cellular Currency S-adenosyl-methionine: Modification of aminoglycosides and nucleic acids
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Streptomycin and spectinomycin are antibiotics that bind to ribosomes and inhibit protein synthesis. Common resistance mechanisms involve enzymatic modification of the two drugs by aminoglycoside nucleotidyltransferases (ANTs). The first part of this thesis covers the structural mechanism of two ANT enzymes. The first is the dual-specificity AadA, belonging to the ANT(3")(9) family, which modifies the 3" position of streptomycin and position 9 of spectinomycin. The second is ANT(9) that only modifies spectinomycin at position 9.

We solved crystal structures of both enzymes, AadA in complex with ATP and streptomycin and ANT(9) with ATP and spectinomycin. The two enzymes show overall structural similarity and both consist of an N-terminal nucleotidyltransferase domain and a C-terminal helical domain. The binding of ATP between the two domains induces a conformational change that allows the drug to bind. The modified hydroxyl groups of both drugs align at similar positions in the active site, even though the drugs are chemically distinct. Comparison of the ANT(9) and AadA structures shows that spectinomycin specificity is explained by the straight α5 helix followed by a short loop in ANT(9) that would clash with larger drug streptomycin. These findings allowed us to explain the substrate recognition of these enzymes and propose a catalytic mechanism.

In the second and third parts of this thesis, I studied two enzymes that use S-adenosyl-methionine (SAM), RlmF in site-specific methylation of ribosomal RNA (rRNA) and Svi3-3 in SAM degradation. SAM is an essential molecule for normal cellular function in all-living cells and termed as a ‘cellular currency’. Knowledge is lacking about the substrate recognition of rRNA methyltransferases and the role of the modifications that they add during ribosome assembly. Here, we identify the residues of RlmF that are critical for binding of the cofactor SAM and the lithium chloride core particle substrate that mimics a 50S ribosome assembly intermediate.In the third part, I present structural and ligand-binding studies of a newly discovered SAM degrading enzyme Svi3-3 from bacteriophage.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2020. , p. 61
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1933
Keywords [en]
Antibiotic resistance, streptomycin, spectinomycin, X-ray crystallography, methyltransferase
National Category
Structural Biology
Research subject
Biology with specialization in Structural Biology
Identifiers
URN: urn:nbn:se:uu:diva-408674ISBN: 978-91-513-0944-6 (print)OAI: oai:DiVA.org:uu-408674DiVA, id: diva2:1423496
Public defence
2020-06-04, A1:111a, Biomedical centrum (BMC), Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2020-05-11 Created: 2020-04-14 Last updated: 2020-05-12
List of papers
1. Structural mechanism of AadA, a dual specificity aminoglycoside adenylyltransferase from Salmonella enterica
Open this publication in new window or tab >>Structural mechanism of AadA, a dual specificity aminoglycoside adenylyltransferase from Salmonella enterica
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2018 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 293, p. 11481-11490Article in journal (Refereed) Published
Abstract [en]

Streptomycin and spectinomycin are antibiotics that bind to the bacterial ribosome and perturb protein synthesis. The clinically most prevalent bacterial resistance mechanism is their chemical modification by aminoglycoside-modifying enzymes such as aminoglycoside nucleotidyltransferases (ANTs). AadA from Salmonella enterica is an aminoglycoside (3’’)(9) adenylyl transferase that O-adenylates position 3” of streptomycin and position 9 of spectinomycin. We previously reported the apo AadA structure with a closed active site. To clarify how AadA binds ATP and its two chemically distinct drug substrates, we here report crystal structures of wildtype AadA complexed with ATP, magnesium, and streptomycin and of an active-site mutant, E87Q, complexed with ATP and streptomycin or the closely related dihydrostreptomycin. These structures revealed that ATP binding induces a conformational change that positions the two domains for drug binding at the interdomain cleft and disclosed the interactions between both domains and the three rings of streptomycin. Spectinomycin docking followed by molecular dynamics simulations suggested that despite the limited structural similarities with streptomycin, spectinomycin makes similar interactions around the modification site, and, in agreement with mutational data, critically interacts with fewer residues. Using structure-guided sequence analyses of ANT(3”)(9) enzymes acting on both substrates and ANT(9) enzymes active only on spectinomycin, we identified sequence determinants for activity on each substrate. We experimentally confirmed that Trp-173 and Asp-178 are essential only for streptomycin resistance. Activity assays indicated that Glu-87 is the catalytic base in AadA and that the non-adenylating E87Q mutant can hydrolyze ATP in the presence of streptomycin.

National Category
Structural Biology Biochemistry and Molecular Biology
Research subject
Biology with specialization in Structural Biology
Identifiers
urn:nbn:se:uu:diva-353761 (URN)10.1074/jbc.RA118.003989 (DOI)000439449700018 ()29871922 (PubMedID)
Funder
Knut and Alice Wallenberg FoundationSwedish Research Council, 2017-03827Swedish Research Council, 2013-05930EU, FP7, Seventh Framework Programme, 283570
Available from: 2018-06-15 Created: 2018-06-15 Last updated: 2020-04-14Bibliographically approved
2. Structural Recognition of Spectinomycin by ResistanceEnzyme ANT(9) from Enterococcus faecalis
Open this publication in new window or tab >>Structural Recognition of Spectinomycin by ResistanceEnzyme ANT(9) from Enterococcus faecalis
(English)In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596Article in journal (Refereed) In press
Abstract [en]

Spectinomycin is a ribosome-binding antibiotic that blocks the translocation

step of translation. A prevalent resistance mechanism is modification of the drug by

aminoglycoside nucleotidyl transferase (ANT) enzymes of the spectinomycin-specific

ANT(9) family or by enzymes of the dual-specificity ANT(3)(9) family, which also acts on

streptomycin. We previously reported the structural mechanism of streptomycin modification

by the ANT(3)(9) AadA from Salmonella enterica. ANT(9) from Enterococcus

faecalis adenylates the 9-hydroxyl of spectinomycin. Here, we present the first structures

of spectinomycin bound to an ANT enzyme. Structures were solved for ANT(9)

in apo- form, in complex with ATP, spectinomycin, and magnesium, or in complex

with only spectinomycin. ANT(9) shows an overall structure similar to that of AadA,

with an N-terminal nucleotidyltransferase domain and a C-terminal -helical domain.

Spectinomycin binds close to the entrance of the interdomain cleft, while ATP is

buried at the bottom. Upon drug binding, the C-terminal domain rotates 14 degrees

to close the cleft, allowing contacts of both domains with the drug. Comparison

with AadA shows that spectinomycin specificity is explained by a straight 5 helix

and a shorter 5-6 loop, which would clash with the larger streptomycin substrate.

In the active site, we observed two magnesium ions, one of them in a previously

unobserved position that may activate the 9-hydroxyl for deprotonation by the catalytic

base Glu-86. The observed binding mode for spectinomycin suggests that spectinamides

and aminomethyl spectinomycins, recent spectinomycin analogues with

expansions in position 4 of the C ring, are also subjected to modification by ANT(9)

and ANT(3)(9) enzymes.

Keywords
Antibiotic resistance
National Category
Structural Biology
Research subject
Biology with specialization in Structural Biology
Identifiers
urn:nbn:se:uu:diva-408616 (URN)10.1128/AAC.00371-20 (DOI)
Available from: 2020-04-09 Created: 2020-04-09 Last updated: 2020-04-14
3. Substrate recognition by 23S RNA methyltransferase RlmF
Open this publication in new window or tab >>Substrate recognition by 23S RNA methyltransferase RlmF
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(English)Manuscript (preprint) (Other academic)
National Category
Structural Biology
Research subject
Biology with specialization in Structural Biology
Identifiers
urn:nbn:se:uu:diva-408617 (URN)
Available from: 2020-04-10 Created: 2020-04-10 Last updated: 2020-04-14
4. Structure of a phage-encoded SAMase enzyme provides insights in substrate binding and mechanism
Open this publication in new window or tab >>Structure of a phage-encoded SAMase enzyme provides insights in substrate binding and mechanism
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(English)Manuscript (preprint) (Other academic)
National Category
Natural Sciences Structural Biology
Identifiers
urn:nbn:se:uu:diva-408808 (URN)
Available from: 2020-04-14 Created: 2020-04-14 Last updated: 2020-04-14

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