I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[12788]
Open Access
Abstract: In the native pathway to therapeutic cannabinoid biosynthesis in Cannabis sativa, the three‐step production of a key intermediate, olivetolic acid, is catalysed by the enzymes tetraketide synthase (TKS; linear tetraketide intermediate production in two stages) and olivetolic acid cyclase (OAC; final C2 → C7 aldol condensation). In the absence of OAC, a nonenzymatic C2 → C7 decarboxylative aldol condensation of the tetraketide intermediate occurs forming olivetol. TKS is a type III polyketide synthase, and the question arises why it is unable to form olivetolic acid directly, but instead forms this unwanted side product. We determined the TKS, CoA complex structure, and performed structurally guided mutagenesis studies to identify potential residues responsible for cyclization pathway discrimination in type III polyketide synthases. Prior studies suggested an ‘aldol switch’ is necessary to allow linear tetraketide intermediate release prior to cyclization, thereby enabling subsequent olivetolic acid production by OAC. However, our studies do not support the presence of a universal or predictable ‘aldol switch’ consensus sequence. Instead, we propose the mode of ordered active site water activation between type III polyketide synthases catalysing different cyclization mechanisms is subtle and homologue‐specific. Our work indicates that subtle structural variations between homologous enzymes can have a major mechanistic impact on the catalytic outcome. This highlights the importance of embedding high‐resolution structural analysis of multiple enzyme homologues with classical site‐directed mutagenesis studies when investigating highly similar enzymes with different mechanistic pathway outcomes.
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Oct 2019
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[8997]
Open Access
Abstract: Natural competence is the term used to describe the uptake of “naked” extracellular DNA by bacteria; it plays a significant role in horizontal genetic exchange. It is associated with type IV pili, and specialized competence pili mediate DNA uptake. Here, we show that the crystal structure of a competence-associated protein from Thermus thermophilus, ComZ, consists of a type II secretion pseudopilin-like domain, with a large β-solenoid domain inserted into the β-sheet of the pilin-like fold. ComZ binds with high affinity to another competence-associated pilin, PilA2, which lies adjacent to the comZ gene in the genome. The crystal structure of PilA2 revealed a similar type II secretion pseudopilin-like fold, with a small subdomain; docking simulations predicted that PilA2 binds between the pseudopilin-like and β-solenoid domains of ComZ. Electrophoretic shift analysis and DNase protection studies were used to show that ComZ alone and the ComZ/PilA2 complex are able to bind DNA. Protection against reductive dimethylation was used in combination with mass spectrometry and site-directed mutagenesis to identify two lysine residues in ComZ which are involved in DNA binding. They are located between the two domains in ComZ, on the opposite side from the predicted PilA2 binding site. These results suggest a model in which PilA2 assists ComZ in forming the competence pilus tip and DNA binds to the side of the fiber. The results demonstrate how a type IV pilin can be adapted to a specific function by domain insertion and provide the first structural insights into a tip-located competence pilin.
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Jun 2019
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[8997, 14509]
Open Access
Abstract: Type IV pili are responsible for a diverse range of functions, including twitching motility and cell adhesion. Assembly of the pilus fiber is driven by a cytoplasmic ATPase: it interacts with an inner membrane complex of biogenesis proteins which, in turn, bind to nascent pilin subunits and mediate fiber assembly. Here we report the structural characterization of the PilF TFP assembly ATPase from Thermus thermophilus. The crystal structure of a recombinant C-terminal fragment of PilF revealed bound, unhydrolysed ATP, although the full length complex was enzymatically active. 3D reconstructions were carried out by single particle cryoelectron microscopy for full length apoprotein PilF and in complex with AMPPNP. The structure forms an hourglass-like shape, with the ATPase domains in one half and the N1 domains in the second half which, we propose, interact with the other pilus biogenesis components. Molecular models for both forms were generated: binding of AMPPNP causes an upward shift of the N1 domains towards the ATPase domains of ~8 Å. We advocate a model in which ATP hydrolysis is linked to displacement of the N1 domains which is associated with lifting pilin subunits out of the inner membrane, and provide the activation energy needed to form the pilus fiber.
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Sep 2018
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[12788]
Abstract: Monoterpenoids offer potential as bio-derived monomer feedstocks for high performance renewable polymers. We describe a biocatalytic route to lactone monomers menthide and dihydrocarvide employing Baeyer-Villiger monooxygenases (BVMOs) from Pseudomonas sp. HI-70 (CPDMO) and Rhodococcus sp. Phi1 (CHMOPhi1) as an alternative to organic synthesis. The regio-selectivity of dihydrocarvide isomer formation was controlled by site-directed mutagenesis of three key active site residues in CHMOPhi1. A combination of crystal structure determination, molecular dynamics simulations and mechanistic modeling using density functional theory (DFT) on a range of models provides insight into the origins of discrimination of wild type (WT) and a variant CHMOPhi1 for producing different regio-isomers of the lactone product. Ring-opening polymerizations of the resultant lactones using mild metal-organic catalysts demonstrate their utility in polymer production. This semi-synthetic approach utilizing a biocatalytic step, non-petroleum feedstocks and mild polymerization catalysts, allows access to known and also to previously unreported and potentially novel lactone monomers and polymers.
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Mar 2018
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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John
Waller
,
Helen
Toogood
,
Vijaykumar
Karuppiah
,
Nicholas John William
Rattray
,
David
Mansell
,
David
Leys
,
John M.
Gardiner
,
Anna
Fryszkowska
,
Syed
Ahmed
,
Rakeshwar
Bandichhor
,
Prashanth Reddy
Gaddameedhi
,
Nigel
Scrutton
Diamond Proposal Number(s):
[12788]
Abstract: Reduction of double bonds of α,β-unsaturated carboxylic acids and esters by ene-reductases remains challenging and it typically requires activation by a second electron-withdrawing moiety, such as a halide or second carboxylate group. We showed that profen precursors, 2-arylpropenoic acids and their esters, were efficiently reduced by Old Yellow Enzymes (OYEs). The XenA and GYE enzymes showed activity towards acids, while a wider range of enzymes were active towards the equivalent methyl esters. Comparative co-crystal structural analysis of profen-bound OYEs highlighted key interactions important in determining substrate binding in a catalytically active conformation. The general utility of ene reductases for the synthesis of (R)-profens was established and this work will now drive future mutagenesis studies to screen for the production of pharmaceutically-active (S)-profens.
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Apr 2017
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Type IV pilins are proteins which form polymers that extend from the surface of the bacterial cell; they are involved in mediating a wide variety of functions, including adhesion, motility and natural competence. Here we describe the determination of the crystal structures of three type IVa pilins proteins from the thermophile Thermus thermophilus. They form part of a cluster of pilus-like proteins within the genome; our results show that one, Tt1222, is very closely related to the main structural type IV pilin, PilA4. The other two, Tt1218 and Tt1219, also adopt canonical pilin-like folds but, interestingly, are most closely related to the structures of the type II secretion system pseudopilins, EpsI/GspI and XcpW/GspJ. GspI and GspJ have been shown to form a complex with another pseudopilin, GspK, and this heterotrimeric complex is known to play a key role in initiating assembly of a pseudopilus which is thought to drive the secretion process. The structural similarity of Tt1218 and Tt1219 to GspI and GspJ suggests that they might work in a similar way, to deliver functions associated with type IV pili in T. thermophilus, such as natural competence.
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Dec 2016
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8997]
Open Access
Abstract: Three enzymes of the Mentha essential oil biosynthetic pathway are highly homologous, namely the ketoreductases (−)-menthone:(−)-menthol reductase and (−)-menthone:(+)-neomenthol reductase, and the “ene” reductase isopiperitenone reductase. We identified a rare catalytic residue substitution in the last two, and performed comparative crystal structure analyses and residue-swapping mutagenesis to investigate whether this determines the reaction outcome. The result was a complete loss of native activity and a switch between ene reduction and ketoreduction. This suggests the importance of a catalytic glutamate vs. tyrosine residue in determining the outcome of the reduction of α,β-unsaturated alkenes, due to the substrate occupying different binding conformations, and possibly also to the relative acidities of the two residues. This simple switch in mechanism by a single amino acid substitution could potentially generate a large number of de novo ene reductases.
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Aug 2016
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7146]
Open Access
Abstract: Neuronal Calcium Sensor-1 (NCS-1) is the primordial member of the Neuronal Calcium Sensor (NCS) family of EF-hand Ca2+-binding proteins. It interacts with both the G-protein coupled receptor (GPCR) dopamine D2 receptor (D2R), regulating its internalisation and surface expression, and the cognate kinases, GRK1 and GRK2. Determination of the crystal structures of Ca2+/NCS-1 alone and in complex with peptides derived from D2R and GRK1 reveals that the differential recognition is facilitated by the conformational flexibility of the C-lobe binding site. We find that two copies of the D2R peptide bind within the hydrophobic crevice on Ca2+/NCS-1, but only one copy of the GRK1 peptide. The different binding modes are made possible by the C-lobe binding site of NCS-1, which adopts alternate conformations in each complex. C-terminal residues S178-V190 act in concert with the flexible EF3/EF4 loop region to effectively form different peptide binding sites. In the Ca2+/NCS-1-D2R peptide complex, the C-terminal region adopts a 310 helix-turn-310 helix, whereas in the GRK1 peptide complex it forms an -helix. Removal of S178-V190 generated a C-terminal truncation mutant which formed a dimer, indicating that the NCS-1 C-terminal region prevents NCS-1 oligomerization. We propose that the flexible nature of the C-terminal region is essential to allow it to modulate its protein binding sites and adapt its conformation to accommodate both ligands. This appears to be driven by the variability of the conformation of the C-lobe binding site, which has ramification for the target specificity and diversity of NCS-1.
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Jul 2015
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Open Access
Abstract: CinA is a widely distributed protein in Gram-positive and Gram-negative bacteria. It is associated with natural competence and is proposed to have a function as an enzyme participating in the pyridine nucleotide cycle, which recycles products formed by non-redox uses of NAD. Here we report the determination of the crystal structure of CinA from Thermus thermophilus, in complex with several ligands. CinA was shown to have both nicotinamide mononucleotide deamidase and ADP-ribose pyrophosphatase activities. The crystal structure shows an unusual asymmetric dimer, with three domains for each chain; the C-terminal domain harbors the nicotinamide mononucleotide deamidase activity, and the structure of a complex with the product nicotinate mononucleotide suggests a mechanism for deamidation. The N-terminal domain belongs to the COG1058 family and is associated with the ADP-ribose pyrophosphatase activity. The asymmetry in the CinA dimer arises from two alternative orientations of the COG1058 domains, only one of which forms a contact with the KH-type domain from the other chain, effectively closing the active site into, we propose, a catalytically competent state. Structures of complexes with Mg2+/ADP-ribose, Mg2+/ATP, and Mg2+/AMP suggest a mechanism for the ADP-ribose pyrophosphatase reaction that involves a rotation of the COG1058 domain dimer as part of the reaction cycle, so that each active site oscillates between open and closed forms, thus promoting catalysis.
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Nov 2014
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Abstract: Type IV pili are long, thin fibers, formed mainly of polymers of a single pilin protein, which are displayed on the surfaces of many bacteria, including several human pathogens. Here, we report three-dimensional reconstructions of the PilMNO inner membrane complex, alone and in complex with pilin protein, through a combination of X-ray crystallography and electron microscopy. PilMNO forms a dimeric T-shaped structure, binding two copies of the pilin protein at its extremities. The results provide a structural model for the way in which pilin is harvested from the inner membrane and made available to other components of the type IV pilus biogenesis machinery.
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Nov 2013
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