I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[1225]
Open Access
Abstract: Trypanosomatids possess glycosome organelles that contain much of the glycolytic machinery, including phosphofructokinase (PFK). We present kinetic and structural data for PFK from three human pathogenic trypanosomatids, illustrating intriguing differences that may reflect evolutionary adaptations to differing ecological niches. The activity of Leishmania PFK – to a much larger extent than Trypanosoma PFK – is reliant on AMP for activity regulation, with 1 mm AMP increasing the L. infantum PFK (LiPFK) kcat/K0.5F6P value by 10‐fold, compared to only a 1.3‐ and 1.4‐fold increase for T. cruzi and T. brucei PFK, respectively. We also show that Leishmania PFK melts at a significantly lower (> 15 °C) temperature than Trypanosoma PFKs and that addition of either AMP or ATP results in a marked stabilization of the protein. Sequence comparisons of Trypanosoma spp. and Leishmania spp. show that divergence of the two genera involved amino acid substitutions that occur in the enzyme’s ‘reaching arms’ and ‘embracing arms’ that determine tetramer stability. The dramatic effects of AMP on Leishmania activity compared with the Trypanosoma PFKs may be explained by differences between the T‐to‐R equilibria for the two families, with the low‐melting Leishmania PFK favouring the flexible inactive T‐state in the absence of AMP. Sequence comparisons along with the enzymatic and structural data presented here also suggest there was a loss of AMP‐dependent regulation in Trypanosoma species rather than gain of this characteristic in Leishmania species and that AMP acts as a key regulator in Leishmania governing the balance between glycolysis and gluconeogenesis.
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Jan 2020
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I03-Macromolecular Crystallography
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Abstract: Coproporphyrin ferrochelatases (CpfCs, EC 4.99.1.9) insert ferrous iron into coproporphyrin III yielding coproheme. CpfCs are utilized by prokaryotic, mainly monoderm (Gram‐positive) bacteria within the recently detected coproporphyrin‐dependent heme biosynthesis pathway. Here we present a comprehensive study on CpfC from Listeria monocytogenes (LmCpfC) including the first crystal structure of a coproheme‐bound CpfC. Comparison of crystal structures of apo‐LmCpfC and coproheme‐LmCpfC allowed identification of structural rearrangements and of amino acids involved in tetrapyrrole macrocycle and Fe2+ binding. Differential scanning calorimetry of apo‐, coproporphyrin III‐ and coproheme‐LmCpfC underline the pronounced non‐covalent interaction of both coproporphyrin and coproheme with the protein (ΔTm = 11 °C compared to apo‐LmCpfC) which includes the propionates (p2, p4, p6, p7) and the amino acids Arg29, Arg45, Tyr46, Ser53 and Tyr124. Furthermore, the thermodynamics and kinetics of coproporphyrin III and coproheme binding to apo‐LmCpfC is presented as well as the kinetics of insertion of ferrous iron into coproporphyrin III‐LmCpfC that immediately leads to formation of ferric coproheme‐LmCpfC (kcat/KM = 4.7 × 105 M‐1 s‐1). We compare the crystal structure of coproheme‐LmCpfC with available structures of CpfCs with artificial tetrapyrrole macrocycles and discuss our data on substrate binding, iron insertion and substrate release in the context of the coproporphyrin‐dependent heme biosynthesis pathway.
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Dec 2019
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
VMXi-Versatile Macromolecular Crystallography in situ
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Diamond Proposal Number(s):
[16818, 19737, 19485]
Open Access
Abstract: Cyclic guanosine 3′,5′‐monophosphate (cGMP) is an intracellular signaling molecule involved in many sensory and developmental processes. Synthesis of cGMP from GTP is catalyzed by guanylate cyclase (GC) in a reaction analogous to cAMP formation by adenylate cyclase (AC). Although detailed structural information is available on the catalytic region of nucleotidyl cyclases (NCs) in various states, these atomic models do not provide a sufficient explanation for the substrate selectivity between GC and AC family members. Detailed structural information on the GC domain in its active conformation is largely missing and no crystal structure of a GTP‐bound wild‐type GC domain has been published to date. Here, we describe the crystal structure of the catalytic domain of rhodopsin‐GC (RhGC) from Catenaria anguillulae in complex with GTP at 1.7 Å resolution. Our study reveals the organization of a eukaryotic GC domain in its active conformation. We observe that the binding mode of the substrate GTP is similar to that of AC–ATP interaction, although surprisingly not all of the interactions predicted to be responsible for base recognition are present. The structure provides insights into potential mechanisms of substrate discrimination and activity regulation that may be common to all class III purine NCs.
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Dec 2019
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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
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Diamond Proposal Number(s):
[8030]
Abstract: Protein trans‐splicing catalyzed by split inteins has increasingly become useful as a protein engineering tool. We solved the 1.0 Å‐resolution crystal structure of a fused variant from the naturally split gp41‐1 intein, previously identified from environmental metagenomic sequence data. The structure of the 125‐residue gp41‐1 intein revealed a compact pseudo‐C2‐symmetry commonly found in the Hedgehog/Intein (HINT) superfamily with extensive charge‐charge interactions between the split N‐ and C‐terminal intein fragments that are common among naturally occurring split inteins. We successfully created orthogonal split inteins by engineering a similar charge network into the same region of a cis‐splicing intein. This strategy could be applicable for creating novel natural‐like split inteins from other, more prevalent cis‐splicing inteins.
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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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