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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I04-1-Macromolecular Crystallography (fixed wavelength)
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Abstract: We describe de novo-designed α-helical barrels (αHBs) that bind and discriminate between lipophilic biologically active molecules. αHBs have five or more α-helices arranged around central hydrophobic channels the diameters of which scale with oligomer state. We show that pentameric, hexameric, and heptameric αHBs bind the environmentally sensitive dye 1,6-diphenylhexatriene (DPH) in the micromolar range and fluoresce. Displacement of the dye is used to report the binding of nonfluorescent molecules: palmitic acid and retinol bind to all three αHBs with submicromolar inhibitor constants; farnesol binds the hexamer and heptamer; but β-carotene binds only the heptamer. A co-crystal structure of the hexamer with farnesol reveals oriented binding in the center of the hydrophobic channel. Charged side chains engineered into the lumen of the heptamer facilitate binding of polar ligands: a glutamate variant binds a cationic variant of DPH, and introducing lysine allows binding of the biosynthetically important farnesol diphosphate.
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Jul 2018
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I03-Macromolecular Crystallography
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Pharit
Kamsri
,
Chayanin
Hanwarinroj
,
Naruedon
Phusi
,
Thimpika
Pornprom
,
Kampanart
Chayajarus
,
Auradee
Punkvang
,
Nitima
Suttipanta
,
Potjanee
Srimanote
,
Khomson
Suttisintong
,
Chomphunuch
Songsiriritthigul
,
Patchreenart
Saparpakorn
,
Supa
Hannongbua
,
Siriluk
Rattanabunyong
,
Supaporn
Seetaha
,
Kiattawee
Choowongkomon
,
Sanya
Sureram
,
Prasat
Kittakoop
,
Poonpilas
Hongmanee
,
Pitak
Santanirand
,
Zhaoqiang
Chen
,
Weiliang
Zhu
,
Rosemary A
Blood
,
Yuiko
Takebayashi
,
Philip
Hinchliffe
,
Adrian J.
Mulholland
,
James
Spencer
,
Pornpan
Pungpo
Diamond Proposal Number(s):
[17212]
Abstract: The enoyl-acyl carrier protein reductase InhA of M. tuberculosis is an attractive, validated target for anti-tuberculosis drug development. Moreover, direct inhibitors of InhA remain effective against InhA variants with mutations associated with isoniazid resistance, offering the potential for activity against MDR isolates. Here, structure based virtual screening supported by biological assays was applied to identify novel InhA inhibitors as potential anti-tuberculosis agents. High-speed Glide SP docking was initially performed against two conformations of InhA differing in the orientation of the active site Tyr158. The resulting hits were filtered for drug-likeness based on Lipinski's rule and avoidance of PAINS-like properties, and finally subjected to Glide XP docking to improve accuracy. Sixteen compounds were identified and selected for in vitro biological assays, of which two (compounds 1 and 7) showed MIC of 12.5 and 25 µg/ml against M. tuberculosis H37Rv, respectively. Inhibition assays against purified recombinant InhA determined IC50 values for these compounds of 0.38 and 0.22 µM, respectively. A crystal structure of the most potent compound, compound 7, bound to InhA revealed the inhibitor to occupy a hydrophobic pocket implicated in binding the aliphatic portions of InhA substrates but distant from the NADH cofactor, i.e. in a site distinct from those occupied by the great majority of known InhA inhibitors. This compound provides an attractive starting template for ligand optimization aimed at discovery of new and effective compounds against M. tuberculosis that act by targeting InhA.
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Dec 2019
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Philip
Hinchliffe
,
Qiu E.
Yang
,
Edward
Portal
,
Tom
Young
,
Hui
Li
,
Catherine L.
Tooke
,
Maria J.
Carvalho
,
Neil G.
Paterson
,
Jurgen
Brem
,
Pannika R.
Niumsup
,
Uttapoln
Tansawai
,
Lei
Lei
,
Mei
Li
,
Zhangqi
Shen
,
Yang
Wang
,
Christopher J.
Schofield
,
Adrian J
Mulholland
,
Jianzhong
Shen
,
Natalie
Fey
,
Timothy R.
Walsh
,
James
Spencer
Diamond Proposal Number(s):
[12342]
Open Access
Abstract: The polymixin colistin is a “last line” antibiotic against extensively-resistant Gram-negative bacteria. Recently, the mcr-1 gene was identified as a plasmid-mediated resistance mechanism in human and animal Enterobacteriaceae, with a wide geographical distribution and many producer strains resistant to multiple other antibiotics. mcr-1 encodes a membrane-bound enzyme catalysing phosphoethanolamine transfer onto bacterial lipid A. Here we present crystal structures revealing the MCR-1 periplasmic, catalytic domain to be a zinc metalloprotein with an alkaline phosphatase/sulphatase fold containing three disulphide bonds. One structure captures a phosphorylated form representing the first intermediate in the transfer reaction. Mutation of residues implicated in zinc or phosphoethanolamine binding, or catalytic activity, restores colistin susceptibility of recombinant E. coli. Zinc deprivation reduces colistin MICs in MCR-1-producing laboratory, environmental, animal and human E. coli. Conversely, over-expression of the disulphide isomerase DsbA increases the colistin MIC of laboratory E. coli. Preliminary density functional theory calculations on cluster models suggest a single zinc ion may be sufficient to support phosphoethanolamine transfer. These data demonstrate the importance of zinc and disulphide bonds to MCR-1 activity, suggest that assays under zinc-limiting conditions represent a route to phenotypic identification of MCR-1 producing E. coli, and identify key features of the likely catalytic mechanism.
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Jan 2017
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[8922, 12342]
Open Access
Abstract: In coiled-coil (CC) protein structures α-helices wrap around one another to form rope-like assemblies. Most natural and designed CCs have two–four helices and cyclic (Cn) or dihedral (Dn) symmetry. Increasingly, CCs with five or more helices are being reported. A subset of these higher-order CCs is of interest as they have accessible central channels that can be functionalised; they are α-helical barrels. These extended cavities are surprising given the drive to maximise buried hydrophobic surfaces during protein folding and assembly in water. Here, we show that α-helical barrels can be maintained by the strategic placement of β-branched aliphatic residues lining the lumen. Otherwise, the structures collapse or adjust to give more-complex multi-helix assemblies without Cn or Dn symmetry. Nonetheless, the structural hallmark of CCs—namely, knobs-into-holes packing of side chains between helices—is maintained leading to classes of CCs hitherto unobserved in nature or accessed by design.
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Oct 2018
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[17212]
Open Access
Abstract: β-Lactamase production is the major β-lactam resistance mechanism in Gram-negative bacteria. β-Lactamase inhibitors (BLIs) efficacious against serine β-lactamase (SBL) producers, especially strains carrying the widely disseminated class A enzymes, are required. Relebactam, a diazabicyclooctane (DBO) BLI is in phase-3 clinical trials in combination with imipenem, for treatment of infections by multi-drug resistant Enterobacteriaceae. We show that relebactam inhibits five clinically-important class A SBLs (despite their differing spectra of activity), representing both chromosomal and plasmid-borne enzymes, i.e. the extended spectrum β-lactamases L2 (inhibition constant 3 μM) and CTX-M-15 (21 μM); and the carbapenemases, KPC-2, -3 and -4 (1 - 5 μM). Against purified class A SBLs, relebactam is an inferior inhibitor compared to the clinically approved DBO avibactam, (9 to 120-fold differences in IC50). Minimum inhibitory concentration assays indicate relebactam potentiates β-lactam (imipenem) activity against KPC-producing Klebsiella pneumoniae with similar potency to avibactam (with ceftazidime). Relebactam is less effective than avibactam in combination with aztreonam against Stenotrophomonas maltophilia K279a. X-ray crystal structures of relebactam bound to CTX-M-15, L2, KPC-2, KPC-3 and KPC-4 reveal its C2 linked piperidine ring can sterically clash with Asn104 (CTX-M-15) or His/Trp105 (L2 and KPCs), rationalizing its poorer inhibition activity compared to avibactam, which has a smaller C2 carboxyamide group. Mass spectrometry and crystallographic data show slow, pH-dependent relebactam desulfation by KPC-2, -3 and -4. This comprehensive comparison of relebactam binding across five clinically-important class A SBLs will inform the design of future DBOs with the aim of improving clinical efficacy of BLI:β-lactam combinations.
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Aug 2019
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8367]
Open Access
Abstract: N-Acetylneuraminic acid lyase (NAL) is a Class I aldolase that catalyzes the reversible condensation of pyruvate with N-acetyl-d-mannosamine (ManNAc) to yield the sialic acid N-acetylneuraminic acid (Neu5Ac). Aldolases are finding increasing use as biocatalysts for the stereospecific synthesis of complex molecules. Incomplete understanding of the mechanism of catalysis in aldolases, however, can hamper development of new enzyme activities and specificities, including control over newly generated stereocenters. In the case of NAL, it is clear that the enzyme catalyzes a Bi-Uni ordered condensation reaction in which pyruvate binds first to the enzyme to form a catalytically important Schiff base. The identity of the residues required for catalysis of the condensation step and the nature of the transition state for this reaction, however, have been a matter of conjecture. In order to address, this we crystallized a Y137A variant of the E. coli NAL in the presence of Neu5Ac. The three-dimensional structure shows a full length sialic acid bound in the active site of subunits A, B, and D, while in subunit C, discontinuous electron density reveals the positions of enzyme-bound pyruvate and ManNAc. These snapshot structures, representative of intermediates in the enzyme catalytic cycle, provided an ideal starting point for QM/MM modeling of the enzymic reaction of carboncarbon bond formation. This revealed that Tyr137 acts as the proton donor to the aldehyde oxygen of ManNAc during the reaction, the activation barrier is dominated by carboncarbon bond formation, and proton transfer from Tyr137 is required to obtain a stable Neu5Ac-Lys165 Schiff base complex. The results also suggested that a triad of residues, Tyr137, Ser47, and Tyr110 from a neighboring subunit, are required to correctly position Tyr137 for its function, and this was confirmed by site-directed mutagenesis. This understanding of the mechanism and geometry of the transition states along the CC bond-forming pathway will allow further development of these enzymes for stereospecific synthesis of new enzyme products.
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Apr 2014
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I24-Microfocus Macromolecular Crystallography
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Emily
Lythell
,
Reynier
Suardíaz
,
Philip
Hinchliffe
,
Chonnikan
Hanpaibool
,
Surawit
Visitsatthawong
,
A. Sofia F.
Oliveira
,
Eric J. M.
Lang
,
Panida
Surawatanawong
,
Vannajan Sanghiran
Lee
,
Thanyada
Rungrotmongkol
,
Natalie
Fey
,
James
Spencer
,
Adrian J.
Mulholland
Diamond Proposal Number(s):
[12342]
Abstract: MCR (mobile colistin resistance) enzymes catalyse phosphoethanolamine (PEA) addition to bacterial lipid A, threatening the “last-resort” antibiotic colistin. Molecular dynamics and density functional theory simulations indicate that monozinc MCR supports PEA transfer to the Thr285 acceptor, positioning MCR as a mono- rather than multinuclear member of the alkaline phosphatase superfamily.
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May 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Vijaykumar
Karuppiah
,
Kara
Ranaghan
,
Nicole G. H.
Leferink
,
Linus O.
Johannissen
,
Muralidharan
Shanmugam
,
Aisling
Ni Cheallaigh
,
Nathan
Bennett
,
Lewis
Kearsey
,
Eriko
Takano
,
John
Gardiner
,
Marc
Van Der Kamp
,
Sam
Hay
,
Adrian J.
Mulholland
,
David
Leys
,
Nigel S.
Scrutton
Diamond Proposal Number(s):
[12788]
Abstract: Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an important class of terpenes and are industrially important as flavours and fragrances. We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthase (bCinS) and show that these are active biocatalysts for monoterpene production using biocatalysis and metabolic engineering platforms. In metabolically engineered monoterpene-producing E. coli strains use of bLinS leads to 300-fold higher linalool production compared with the corresponding plant monoterpene synthase. With bCinS, 1,8-cineole is produced with 96% purity compared to 67% from plant species. Structures of bLinS and bCinS, and their complexes with fluorinated substrate analogues, show that these bacterial monoterpene synthases are similar to previously characterised sesquiterpene synthases. Molecular dynamics simulations suggest that these monoterpene synthases do not undergo large-scale conformational changes during the reaction cycle making them attractive targets for structured-based protein engineering to expand the catalytic scope of these enzymes towards alternative monoterpene scaffolds. Comparison of the bLinS and bCinS structures indicates how their active sites steer reactive carbocation intermediates to the desired acyclic linalool (bLinS) or bicyclic 1,8-cineole (bCinS) products. The work reported here provides the analysis of structures for this important class of monoterpene synthase. This should now guide exploitation of the bacterial enzymes as gateway biocatalysts for the production of other monoterpenes and monoterpenoids.
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Aug 2017
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I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[8922]
Abstract: The Diels–Alder reaction, a [4 + 2] cycloaddition of a conjugated diene to a dienophile, is one of the most powerful reactions in synthetic chemistry. Biocatalysts capable of unlocking new and efficient Diels–Alder reactions would have major impact. Here we present a molecular-level description of the reaction mechanism of the spirotetronate cyclase AbyU, an enzyme shown here to be a bona fide natural Diels–Alderase. Using enzyme assays, X-ray crystal structures, and simulations of the reaction in the enzyme, we reveal how linear substrate chains are contorted within the AbyU active site to facilitate a transannular pericyclic reaction. This study provides compelling evidence for the existence of a natural enzyme evolved to catalyze a Diels–Alder reaction and shows how catalysis is achieved.
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May 2016
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