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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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[10071]
Open Access
Abstract: Burkholderia pseudomallei is a serious, difficult to treat Gram‐negative pathogen and an increase in the occurrence of drug‐resistant strains has been detected. We have directed efforts to identify and to evaluate potential drug targets relevant to treatment of infection by B. pseudomallei. We have selected and characterised the essential enzyme d‐alanine‐d‐alanine ligase (BpDdl), required for the ATP‐assisted biosynthesis of a peptidoglycan precursor. A recombinant supply of protein supported high‐resolution crystallographic and biophysical studies with ligands (AMP and AMP+d‐Ala‐d‐Ala), and comparisons with orthologues enzymes suggest a ligand‐induced conformational change occurring that might be relevant to the catalytic cycle. The detailed biochemical characterisation of the enzyme, development and optimisation of ligand binding assays supported the search for novel inhibitors by screening of selected compound libraries. In a similar manner to that observed previously in other studies, we note a paucity of hits that are worth follow‐up and then in combination with a computational analysis of the active site, we conclude that this ligase represents a difficult target for drug discovery. Nevertheless, our reagents, protocols and data can underpin future efforts exploiting more diverse chemical libraries and structure‐based approaches.
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Jul 2019
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[11740, 15991]
Open Access
Abstract: Methylation is an underpinning process of life and provides control for biological
processes such as DNA synthesis, cell growth, and apoptosis. Methionine
adenosyltransferases (MAT) produce the cellular methyl donor, S-Adenosylmethionine
(SAMe). Dysregulation of SAMe level is a relevant event in many
diseases, including cancers such as hepatocellular carcinoma and colon cancer.
In addition, mutation of Arg264 in MATa1 causes isolated persistent hypermethioninemia,
which is characterized by low activity of the enzyme in liver and
high level of plasma methionine. In mammals, MATa1/a2 and MATbV1/V2
are the catalytic and the major form of regulatory subunits, respectively. A gating
loop comprising residues 113–131 is located beside the active site of catalytic
subunits (MATa1/a2) and provides controlled access to the active site. Here, we
provide evidence of how the gating loop facilitates the catalysis and define some
of the key elements that control the catalytic efficiency. Mutation of several residues
of MATa2 including Gln113, Ser114, and Arg264 lead to partial or total
loss of enzymatic activity, demonstrating their critical role in catalysis. The enzymatic
activity of the mutated enzymes is restored to varying degrees upon complex
formation with MATbV1 or MATbV2, endorsing its role as an allosteric
regulator of MATa2 in response to the levels of methionine or SAMe. Finally,
the protein–protein interacting surface formed in MATa2:MATb complexes is
explored to demonstrate that several quinolone-based compounds modulate the
activity of MATa2 and its mutants, providing a rational for chemical design/intervention
responsive to the level of SAMe in the cellular environment.
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Mar 2019
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[17221]
Abstract: Frataxins form an interesting family of iron‐binding proteins with an almost unique fold and are highly conserved from bacteria to primates. They have a pivotal role in iron‐sulfur cluster biogenesis as regulators of the rates of cluster formation, as it is testified by the fact that frataxin absence is incompatible with life and reduced levels of the protein lead to the recessive neurodegenerative disease Friedreich's ataxia. Despite its importance, the structure of frataxin has been solved only from relatively few species. Here, we discuss the X‐ray structure of frataxin from the thermophilic fungus Chaetomium thermophilum, and the characterization of its interactions and dynamics in solution. We show that this eukaryotic frataxin has an unusual variation of the classical frataxin fold: the last helix is shorter than in other frataxins which results in a less symmetrical and compact structure. The stability of this protein is comparable to that of human frataxin, currently the most stable amongst the frataxin orthologues. We also characterized the iron‐binding mode of C. thermophilum frataxin and demonstrated that it binds it through a semi‐conserved negatively charged ridge on the first helix and beta‐strand. Moreover, this frataxin is also able to bind the bacterial ortholog of the desulfurase, which is central in iron‐sulfur cluster synthesis, and act as its inhibitor.
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Jan 2019
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14198, 15369, 16452, 17251]
Abstract: Energy metabolism in the diamondback moth Plutella xylostella is facilitated by trehalase, an enzyme which assists in trehalose hydrolysis, from the predominant gut bacterium Enterobacter cloacae. We report the biochemical and structural characterization of recombinant trehalase from Enterobacter cloacae (Px_EclTre). Px_EclTre showed KM of 1.47 (±0.05) mM, kcat of 6254.72 min−1 and Vmax 0.2 (±0.002) mM/min at 55 °C and acidic pH. Crystal structures of Px_EclTre were determined in the ligand‐free form and bound to the inhibitor Validoxylamine A. The crystal structure of the ligand‐free form, unavailable until now for any other bacterial trehalases, enabled us to delineate the conformational changes accompanying ligand binding in trehalases. Multiple salt bridges were identified that potentially facilitated closure of a hood over the substrate‐binding site. A cluster of five tryptophans lined the ‐1 substrate binding subsite, interacted with crucial active site residues, and contributed to both trehalase activity and stability. The importance of these residues in enzyme activity was further validated by mutagenesis studies. Many of these identified residues form part of signature motifs and other conserved sequences in trehalases. The structure analysis thus led to the assignment of the functional role to these conserved residues. This information can be further explored for the design of effective inhibitors against trehalases.
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Jan 2019
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Ramya
Salimraj
,
Philip
Hinchliffe
,
Magda
Kosmopoulou
,
Jonathan M.
Tyrrell
,
Jurgen
Brem
,
Sander S.
Van Berkel
,
Anil
Verma
,
Raymond J.
Owens
,
Michael A.
Mcdonough
,
Timothy R.
Walsh
,
Christopher J.
Schofield
,
James
Spencer
Diamond Proposal Number(s):
[313]
Abstract: Metallo‐β‐Lactamases (MBLs) protect bacteria from almost all β‐lactam antibiotics. VIM enzymes are among the most clinically important MBLs, with VIM‐1 increasing in carbapenem‐resistant Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae) that are amongst the hardest bacterial pathogens to treat. VIM enzymes display sequence variation at residues (224 and 228) that in related MBLs are conserved and participate in substrate binding. How they accommodate this variability, while retaining catalytic efficiency against a broad substrate range, has remained unclear. Here we present crystal structures of VIM‐1 and its complexes with a substrate‐mimicking thioenolate inhibitor, ML302F, that restores meropenem activity against a range of VIM‐1 producing clinical strains, and the hydrolysed product of the carbapenem meropenem. Comparison of these two structures identifies a water‐mediated hydrogen bond, between the carboxylate group of substrate/inhibitor and the backbone carbonyl of the active site zinc ligand Cys221, that is common to both complexes. Structural comparisons show that the responsible Cys221‐bound water is observed in all known VIM structures, participates in carboxylate binding with other inhibitor classes, and thus effectively replicates the role of the conserved Lys224 in analogous complexes with other MBLs. These results provide a mechanism for substrate binding that permits the variation at positions 224 and 228 that is a hallmark of VIM MBLs.
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Nov 2018
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9495]
Abstract: The identification of new strategies to fight bacterial infections in view of the spread of multiple resistance to antibiotics has become mandatory. It has been demonstrated that several bacteria develop poly‐γ‐glutamic acid (γ‐PGA) capsules as a protection from external insults and/or host defence systems. Among the pathogens that shield themselves in these capsules are B. anthracis, F. tularensis and several Staphylococcus strains. These are important pathogens with a profound influence on human health. The recently characterised γ‐PGA hydrolases, which can dismantle the γ‐PGA‐capsules, are an attractive new direction that can offer real hope for the development of alternatives to antibiotics, particularly in cases of multidrug resistant bacteria. We have characterised in detail the cleaving mechanism and stereospecificity of the enzyme PghL (previously named YndL) from B. subtilis encoded by a gene of phagic origin and dramatically efficient in degrading the long polymeric chains of γ‐PGA. We used X‐ray crystallography to solve the three‐dimensional structures of the enzyme in its zinc‐free, zinc‐bound and complexed forms. The protein crystallised with a γ‐PGA hexapeptide substrate and thus reveals details of the interaction which could explain the stereospecificity observed and give hints on the catalytic mechanism of this class of hydrolytic enzymes.
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Nov 2018
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14692]
Abstract: The identification of enzymes responsible for oxidation of lignin in lignin‐degrading bacteria is of interest for biotechnological valorization of lignin to renewable chemical products. The genome sequences of two lignin‐degrading bacteria, Ochrobactrum sp., and Paenibacillus sp., contain no B‐type DyP peroxidases implicated in lignin degradation in other bacteria, but contain putative multicopper oxidase genes. Multi‐copper oxidase CueO from Ochrobactrum sp. was expressed and reconstituted as a recombinant laccase‐like enzyme, and kinetically characterized. Ochrobactrum CueO shows activity for oxidation of β‐aryl ether and biphenyl lignin dimer model compounds, generating oxidized dimeric products, and shows activity for oxidation of Ca‐lignosulfonate, generating vanillic acid as a low molecular weight product. The crystal structure of Ochrobactrum CueO (OcCueO) has been determined at 1.1 Å resolution (PDB: 6EVG), showing a four‐coordinate mononuclear type I copper center with ligands His495, His434 and Cys490 with Met500 as an axial ligand, similar to that of Escherichia coli CueO and bacterial azurin proteins, whereas fungal laccase enzymes contain a three‐coordinate type I copper metal center. A trinuclear type 2/3 copper cluster was modeled into the active site, showing similar structure to E. coli CueO and fungal laccases, and three solvent channels leading to the active site. Site‐directed mutagenesis was carried out on amino acid residues found in the solvent channels, indicating the importance for residues Asp102, Gly103, Arg221, Arg223, and Asp462 for catalytic activity. The work identifies a new bacterial multicopper enzyme with activity for lignin oxidation, and implicates a role for bacterial laccase‐like multicopper oxidases in some lignin‐degrading bacteria.
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Mar 2018
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[12342]
Abstract: Angiotensin-1 converting enzyme (ACE) is a zinc metallopeptidase that consists of two homologous catalytic domains (known as nACE and cACE) with different substrate specificities. Based on kinetic studies it was previously reported that sampatrilat, a tight-binding inhibitor of ACE, Ki =13.8 nM and 171.9 nM for cACE and nACE respectively [Sharma et al., Journal of Chemical Information and Modeling (2016), 56, 2486-2494] was 12.4-fold more selective for cACE. In addition, samAsp, in which an aspartate group replaces the sampatrilat lysine, was found to be a non-specific and lower micromolar affinity inhibitor. Here we report a detailed three-dimensional structural analysis of sampatrilat and samAsp binding to ACE using high resolution crystal structures elucidated by X-ray crystallography, which provides a molecular basis for differences in inhibitor affinity and selectivity for nACE and cACE. The structures show that the specificity of sampatrilat can be explained by increased hydrophobic interactions and a H-bond from Glu403 of cACE with the lysine side chain of sampatrilat that are not observed in nACE. In addition, the structures clearly show a significantly greater number of hydrophilic and hydrophobic interactions with sampatrilat compared to samAsp in both cACE and nACE consistent with the difference in affinities. Our findings provide new experimental insights into ligand binding at the active site pockets that are important for the design of highly specific domain selective inhibitors of ACE.
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Feb 2018
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I04-Macromolecular Crystallography
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Filipa
Mota
,
Constantina
Fotinou
,
Rohini
Rhana
,
A. W.
Edith Chan
,
Tamas
Yelland
,
Mohamed T.
Arooz
,
Andrew P.
O'leary
,
Jennie
Hutton
,
Paul
Frankel
,
Ian
Zachary
,
David
Selwood
,
Snezana
Djordjevic
Diamond Proposal Number(s):
[12305]
Open Access
Abstract: Neuropilin-1 (NRP1) is a transmembrane co-receptor involved in binding interactions with variety of ligands and receptors, including receptor tyrosine kinases. Expression of NRP1 in several cancers correlates with cancer stages and poor prognosis. Thus, NRP1 has been considered a therapeutic target and is the focus of multiple drug discovery initiatives. Vascular endothelial growth factor (VEGF) binds to the b1 domain of NRP1 through interactions between the C-terminal arginine of VEGF and residues in the NRP1 binding site including Tyr297, Tyr353, Asp320, Ser346, and Thr349. We obtained several complexes of the synthetic ligands and the NRP1-b1 domain and used X-ray crystallography and computational methods to analyze atomic details and hydration profile of this binding site. We observed side chain flexibility for Tyr297 and Asp320 in the 6 new high resolution crystal structures of arginine analogues bound to NRP1. In addition, we identified conserved water molecules in binding site regions which can be targeted for drug design. The computational prediction of the VEGF ligand-binding site hydration map of NRP1 was in agreement with the experimentally-derived, conserved hydration structure. Displacement of certain conserved water molecules by a ligand's functional groups may contribute to binding affinity, whilst other water molecules perform as protein - ligand bridges. Our report provides a comprehensive description of the binding site for the peptidic ligands’ C-terminal arginines in the b1 domain of NRP1, highlights the importance of conserved structural waters in drug design, and validates the utility of the computational hydration map prediction method in the context of neuropilin.
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Feb 2018
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