18 research outputs found
Structure of NS1A effector domain from the influenza A/Udorn/72 virus
The structure of the effector domain of the influenza protein NS1, a validated antiviral drug target, has been solved in two space groups
Promiscuous Partitioning of a Covalent Intermediate Common in the Pentein Superfamily
SummaryMany enzymes in the pentein superfamily use a transient covalent intermediate in their catalytic mechanisms. Here we trap and determine the structure of a stable covalent adduct that mimics this intermediate using a mutant dimethylarginine dimethylaminohydrolase and an alternative substrate. The interactions observed between the enzyme and trapped adduct suggest an altered angle of attack between the nucleophiles of the first and second half-reactions of normal catalysis. The stable covalent adduct is also capable of further reaction. Addition of imidazole rescues the original hydrolytic activity. Notably, addition of other amines instead yields substituted arginine products, which arise from partitioning of the intermediate into the evolutionarily related amidinotransferase reaction pathway. The enzyme provides both selectivity and catalysis for the amidinotransferase reaction, underscoring commonalities among the reaction pathways in this mechanistically diverse enzyme superfamily. The promiscuous partitioning of this intermediate may also help to illuminate the evolutionary history of these enzymes
Crystallization And Preliminary X-Ray Analysis Of A Chitinase From The Fungal Pathogen Coccidioides Immitis
Chitinase is necessary for fungal growth and cell division and, therefore, is an ideal target for the design of inhibitors which may act as antifungal agents. A chitinase from the fungal pathogen Coccidioides immitis has been expressed as a fusion protein with gluathione-S-transferase (GST), which aids in purification. After cleavage from GST, chitinase was crystallized from 30% PEG 4000 in 0.1 M sodium acetate pH 4.6. The crystals have a tetragonal crystal lattice and belong to space group P4(1)2(1)2 or P4(3)2(1)2 and diffract to 2.2 Angstrom resolution. The unit-cell parameters are a = b = 91.2, c = 95.4 Angstrom; there is only one chitinase molecule in the asymmetric unit.National Institutes of Health GM 30048National Science Foundation MCB-9601096Foundation for ResearchWelch FoundationMolecular Bioscience
Using X-Ray Crystallography of the Asp55Asn Mutant of the Phosphatidylcholine-Preferring Phospholipase C from Bacillus Cereus to Support the Mechanistic Role of Asp55 As the General Base
Because mutations of the ionizable Asp at position 55 of the phosphatidylcholine preferring phospholipase C from Bacillus cereus (PLCBc) to a non-ionizable Asn generate a mutant enzyme (D55N) with 104-fold lower catalytic activity than the wild-type enzyme, we tentatively identified Asp55 as the general base for the enzymatic reaction. To eliminate the alternate possibility that Asp55 is a structurally important amino acid, the X-ray structures of unbound D55N and complexes of D55N with two non-hydrolyzable substrate analogues have been solved and refined to 2.0, 2.0, and 2.3Ã…, respectively. The structures of unbound wild-type PLCBc and a wild-type PLCBc-complex with a non-hydrolyzable substrate analogue do not change significantly as a result of replacing Asp55 with Asn. These observations demonstrate that Asp55 is not critical for the structural integrity of the enzyme and support the hypothesis that Asp55 is the general base in the PLCBc-catalyzed hydrolysis of phospholipids
The structure of chitinases and prospects for structure-based drug design
Abstract: Many fungi, including pathogenic strains, require proper chitin metabolism to assure normal cell wall replication. Chitinase hydrolyzes chitin; inhibition of endogenous chitinases or application of extracellular chitinases can disrupt fungal division. It is possible that chitinase inhibitors could be used as antifungal agents. We have solved the X-ray structure of a class I1 chitinase from barley and proposed a mechanism of action. The enzyme has a structural core similar to lysozyme and probably acts in a similar catalytic manner. The enzyme structure can, in principle, be used to identify small molecules that will bind avidly to the active site and act as inhibitors. Those inhibitors that embody transition state geometry are likely to be particularly effective. Key words: chitinase, mechanism of action, drug design. RCsumk : Plusieurs champignons, incluant des souches pathogtnes, ont besoin d'un mttabolisme de la chitine approprit pour assurer une reproduction normale de la paroi cellulaire. La chitinase hydrolyse la chitine; l'inhibition des chitinases endogknes ou l'application de chitinases extracellulaires peut bloquer la division fongique. I1 est possible que des inhibiteurs de la chitinase puissent Ctre utilists comme substances antifongiques. Les auteurs ont dtchiffrt la structure, aux rayons X, d'une chitinase de classe I1 chez I'orge, et proposent un mtcanisme d'action. L'enzyme posstde une structure centrale semblable i celle du lyzozyme et agit probablement d'une fa~on catalytique comparable. La structure de l'enzyme peut, en principe, Ctre utiliste pour identifier de petites moltcules qui s'attachent fortement au site actif pour agir comme inhibiteurs. Ces inhibiteurs, qui montrent un ttat gtomttrique de transition, ont des chances d'Ctre particulikrement efficaces
Developing Dual and Specific Inhibitors of Dimethylarginine Dimethylaminohydrolase-1 and Nitric Oxide Synthase: Toward a Targeted Polypharmacology To Control Nitric Oxide
Dissection, Optimization, and Structural Analysis of a Covalent Irreversible DDAH1 Inhibitor
Inhibitors
of the human enzyme dimethylarginine dimethylaminohydrolase-1
(DDAH1) can control endogenous nitric oxide production. A time-dependent
covalent inactivator of DDAH1, <i>N</i><sup>5</sup>-(1-imino-2-chloroethyl)-l-ornithine (<i>K</i><sub>I</sub> = 1.3 μM, <i>k</i><sub>inact</sub> = 0.34 min<sup>–1</sup>), was conceptually
dissected into two fragments and each characterized separately: l-norvaline (<i>K</i><sub>i</sub> = 470 μM)
and 2-chloroacetamidine (<i>K</i><sub>I</sub> = 310 μM, <i>k</i><sub>inact</sub> = 4.0 min<sup>–1</sup>). This analysis
suggested that the two fragments were not linked in a manner that
allows either to reach full affinity or reactivity, prompting the
synthesis and characterization of three analogues: two that mimic
the dimethylation status of the substrate, <i>N</i><sup>5</sup>-(1-imino-2-chloroisopropyl)-l-ornithine (<i>k</i><sub>inact</sub><i>/K</i><sub>I</sub> = 208 M<sup>–1</sup> s<sup>–1</sup>) and <i>N</i><sup>5</sup>-(1-imino-2-chlorisopropyl)-l-lysine (<i>k</i><sub>inact</sub><i>/K</i><sub>I</sub> = 440 M<sup>–1</sup> s<sup>–1</sup>), and one that lengthens the linker beyond
that found in the substrate, <i>N</i><sup>5</sup>-(1-imino-2-chloroethyl)-l-lysine (Cl-NIL, <i>K</i><sub>I</sub> = 0.19 μM, <i>k</i><sub>inact</sub> = 0.22 min<sup>–1</sup>). Cl-NIL
is one of the most potent inhibitors reported for DDAH1, inactivates
with a second order rate constant (1.9 × 10<sup>4</sup> M<sup>–1</sup> s<sup>–1</sup>) larger than the catalytic
efficiency of DDAH1 for its endogenous substrate (1.6 × 10<sup>2</sup> M<sup>–1</sup> s<sup>–1</sup>), and has a partition
ratio of 1 with a >100 000-fold selectivity for DDAH1 over
arginase. An activity-based protein-profiling probe is used to show
inhibition of DDAH1 within cultured HEK293T cells (IC<sub>50</sub> = 10 μM) with cytotoxicity appearing only at higher concentrations
(ED<sub>50</sub> = 118 μM). A 1.91 Å resolution X-ray crystal
structure reveals specific interactions made with DDAH1 upon covalent
inactivation by Cl-NIL. Dissecting a covalent inactivator and analysis
of its constituent fragments proved useful for the design and optimization
of this potent and effective DDAH1 inhibitor