| 1. |
- Durall de la Fuente, Claudia, et al.
(författare)
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Oligomerization and characteristics of phosphoenolpyruvate carboxylase in Synechococcus PCC 7002
- 2020
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Phosphoenolpyruvate carboxylase (PEPc) is an essential enzyme in plants. A photosynthetic form is present both as dimer and tetramer in C4 and CAM metabolism. Additionally, non-photosynthetic PEPcs are also present. The single, non-photosynthetic PEPc of the unicellular cyanobacterium Synechococcus PCC 7002 (Synechococcus), involved in the TCA cycle, was examined. Using size exclusion chromatography (SEC) and small angle X-ray scattering (SAXS), we observed that PEPc in Synechococcus exists as both a dimer and a tetramer. This is the first demonstration of two different oligomerization states of a non-photosynthetic PEPc. High concentration of Mg2+, the substrate PEP and a combination of low concentration of Mg2+ and HCO3− induced the tetramer form of the carboxylase. Using SEC-SAXS analysis, we showed that the oligomerization state of the carboxylase is concentration dependent and that, among the available crystal structures of PEPc, the scattering profile of PEPc of Synechococcus agrees best with the structure of PEPc from Escherichia coli. In addition, the kinetics of the tetramer purified in presence of Mg2+ using SEC, and of the mixed population purified in presence of Mg2+ using a Strep-tagged column were examined. Moreover, the enzyme showed interesting allosteric regulation, being activated by succinate and inhibited by glutamine, and not affected by either malate, 2-oxoglutarate, aspartic acid or citric acid.
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| 2. |
- Guo, Xiaohu, et al.
(författare)
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Structure and mechanism of a phage-encoded SAM lyase revises catalytic function of enzyme family
- 2021
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Ingår i: eLIFE. - : eLife Sciences Publications Ltd. - 2050-084X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- The first S-adenosyl methionine (SAM) degrading enzyme (SAMase) was discovered in bacteriophage T3, as a counter-defense against the bacterial restriction-modification system, and annotated as a SAM hydrolase forming 5’-methyl-thioadenosine (MTA) and L-homoserine. From environmental phages, we recently discovered three SAMases with barely detectable sequence similarity to T3 SAMase and without homology to proteins of known structure. Here, we present the very first phage SAMase structures, in complex with a substrate analogue and the product MTA. The structure shows a trimer of alpha–beta sandwiches similar to the GlnB-like superfamily, with active sites formed at the trimer interfaces. Quantum-mechanical calculations, thin-layer chromatography, and nuclear magnetic resonance spectroscopy demonstrate that this family of enzymes are not hydrolases but lyases forming MTA and L-homoserine lactone in a unimolecular reaction mechanism. Sequence analysis and in vitro and in vivo mutagenesis support that T3 SAMase belongs to the same structural family and utilizes the same reaction mechanism.
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| 3. |
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| 4. |
- Kanchugal Puttaswamy, Sandesh, et al.
(författare)
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Structural Recognition of Spectinomycin by Resistance Enzyme ANT(9) from Enterococcus faecalis
- 2020
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Ingår i: Antimicrobial Agents and Chemotherapy. - : American Society for Microbiology. - 0066-4804 .- 1098-6596. ; 64:6
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Tidskriftsartikel (refereegranskat)abstract
- 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 alpha-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 alpha(5) helix and a shorter alpha(5)-alpha(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.
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| 5. |
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| 6. |
- Larsson, Daniel S D, et al.
(författare)
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Structural Consequences of Deproteinating the 50S Ribosome
- 2022
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Ingår i: Biomolecules. - : MDPI. - 2218-273X. ; 12:11
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Tidskriftsartikel (refereegranskat)abstract
- Ribosomes are complex ribonucleoprotein particles. Purified 50S ribosomes subjected to high-salt wash, removing a subset of ribosomal proteins (r-proteins), were shown as competent for in vitro assembly into functional 50S subunits. Here, we used cryo-EM to determine the structures of such LiCl core particles derived from E. coli 50S subunits. A wide range of complexes with large variations in the extent of the ordered 23S rRNA and the occupancy of r-proteins were resolved to between 2.8 angstrom and 9 angstrom resolution. Many of these particles showed high similarity to in vivo and in vitro assembly intermediates, supporting the inherent stability or metastability of these states. Similar to states in early ribosome assembly, the main class showed an ordered density for the particle base around the exit tunnel, with domain V and the 3 '-half of domain IV disordered. In addition, smaller core particles were discovered, where either domain II or IV was unfolded. Our data support a multi-pathway in vitro disassembly process, similar but reverse to assembly. Dependencies between complex tertiary RNA structures and RNA-protein interactions were observed, where protein extensions dissociated before the globular domains. We observed the formation of a non-native RNA structure upon protein dissociation, demonstrating that r-proteins stabilize native RNA structures and prevent non-native interactions also after folding.
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| 7. |
- Rajkovic, Andrei, et al.
(författare)
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Amino acid substitutions in human growth hormone affect secondary structure and receptor binding
- 2023
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Ingår i: PLOS ONE. - : PUBLIC LIBRARY SCIENCE. - 1932-6203. ; 18:3
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Tidskriftsartikel (refereegranskat)abstract
- The interaction between human Growth Hormone (hGH) and hGH Receptor (hGHR) has basic relevance to cancer and growth disorders, and hGH is the scaffold for Pegvisomant, an anti-acromegaly therapeutic. For the latter reason, hGH has been extensively engineered by early workers to improve binding and other properties. We are particularly interested in E174 which belongs to the hGH zinc-binding triad; the substitution E174A is known to significantly increase binding, but to now no explanation has been offered. We generated this and several computationally-selected single-residue substitutions at the hGHR-binding site of hGH. We find that, while many successfully slow down dissociation of the hGH-hGHR complex once bound, they also slow down the association of hGH to hGHR. The E174A substitution induces a change in the Circular Dichroism spectrum that suggests the appearance of coiled-coiling. Here we show that E174A increases affinity of hGH against hGHR because the off-rate is slowed down more than the on-rate. For E174Y (and certain mutations at other sites) the slowdown in on-rate was greater than that of the off-rate, leading to decreased affinity. The results point to a link between structure, zinc binding, and hGHR-binding affinity in hGH.
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| 8. |
- Stern, Ana Laura, et al.
(författare)
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Structural mechanism of AadA, a dual specificity aminoglycoside adenylyltransferase from Salmonella enterica
- 2018
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 293, s. 11481-11490
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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