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13 items found (0 items not approved), displaying 11 to 13.[First/Prev] 1, 2 [Next/Last]

Instruments / Technical Area	Publication Details	Published
Krios II-Titan Krios II at Diamond	Cryo-EM and directed evolution reveal how Arabidopsis nitrilase specificity is influenced by its quaternary structure Andani Mulelu , Angela Kirykowicz , Jeremy Woodward Communications Biology 2 DOI: 10.1038/s42003-019-0505-4 PMID: 31341959 Diamond Proposal Number(s): [20975] Open Access Show Abstract ▼ Abstract: Nitrilases are helical enzymes that convert nitriles to acids and/or amides. All plants have a nitrilase 4 homolog specific for ß-cyanoalanine, while in some plants neofunctionalization has produced nitrilases with altered specificity. Plant nitrilase substrate size and specificity correlate with helical twist, but molecular details of this relationship are lacking. Here we determine, to our knowledge, the first close-to-atomic resolution (3.4 Å) cryo-EM structure of an active helical nitrilase, the nitrilase 4 from Arabidopsis thaliana. We apply site-saturation mutagenesis directed evolution to three residues (R95, S224, and L169) and generate a mutant with an altered helical twist that accepts substrates not catalyzed by known plant nitrilases. We reveal that a loop between α2 and α3 limits the length of the binding pocket and propose that it shifts position as a function of helical twist. These insights will allow us to start designing nitrilases for chemoenzymatic synthesis.	Jul 2019
I24-Microfocus Macromolecular Crystallography	Structure of Mycobacterium tuberculosis phosphatidylinositol phosphate synthase reveals mechanism of substrate binding and metal catalysis Kristine Grave , Matthew D. Bennett , Martin Högbom Communications Biology 2 DOI: 10.1038/s42003-019-0427-1 Diamond Proposal Number(s): [11265] Open Access Show Abstract ▼ Abstract: Tuberculosis causes over one million yearly deaths, and drug resistance is rapidly developing. Mycobacterium tuberculosis phosphatidylinositol phosphate synthase (PgsA1) is an integral membrane enzyme involved in biosynthesis of inositol-derived phospholipids required for formation of the mycobacterial cell wall, and a potential drug target. Here we present three crystal structures of M. tuberculosis PgsA1: in absence of substrates (2.9 Å), in complex with Mn2+ and citrate (1.9 Å), and with the CDP-DAG substrate (1.8 Å). The structures reveal atomic details of substrate binding as well as coordination and dynamics of the catalytic metal site. In addition, molecular docking supported by mutagenesis indicate a binding mode for the second substrate, D-myo-inositol-3-phosphate. Together, the data describe the structural basis for M. tuberculosis phosphatidylinositol phosphate synthesis and suggest a refined general catalytic mechanism—including a substrate-induced carboxylate shift—for Class I CDP-alcohol phosphotransferases, enzymes essential for phospholipid biosynthesis in all domains of life.	May 2019
I02-Macromolecular Crystallography I04-Macromolecular Crystallography	15-deoxy-Δ12,14-Prostaglandin J2 inhibits human soluble epoxide hydrolase by a dual orthosteric and allosteric mechanism Giancarlo Abis , Rebecca L. Charles , Jolanta Kopec , Wyatt W. Yue , R. Andrew Atkinson , Tam T. T. Bui , Steven Lynham , Simona Popova , Yin-biao Sun , Franca Fraternali , Philip Eaton , Maria R. Conte Communications Biology 2 DOI: 10.1038/s42003-019-0426-2 Diamond Proposal Number(s): [13597, 10619] Open Access Show Abstract ▼ Abstract: Human soluble epoxide hydrolase (hsEH) is an enzyme responsible for the inactivation of bioactive epoxy fatty acids, and its inhibition is emerging as a promising therapeutical strategy to target hypertension, cardiovascular disease, pain and insulin sensitivity. Here, we uncover the molecular bases of hsEH inhibition mediated by the endogenous 15-deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2). Our data reveal a dual inhibitory mechanism, whereby hsEH can be inhibited by reversible docking of 15d-PGJ2 in the catalytic pocket, as well as by covalent locking of the same compound onto cysteine residues C423 and C522, remote to the active site. Biophysical characterisations allied with in silico investigations indicate that the covalent modification of the reactive cysteines may be part of a hitherto undiscovered allosteric regulatory mechanism of the enzyme. This study provides insights into the molecular modes of inhibition of hsEH epoxy-hydrolytic activity and paves the way for the development of new allosteric inhibitors.	May 2019

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