scholarly journals Evidence that cysteine-166 is the active-site nucleophile of Pseudomonas aeruginosa amidase: crystallization and preliminary X-ray diffraction analysis of the enzyme

1999 ◽  
Vol 340 (3) ◽  
pp. 711-714 ◽  
Author(s):  
Sebastien FARNAUD ◽  
Renée TATA ◽  
Maninder K. SOHI ◽  
Tommy WAN ◽  
Paul R. BROWN ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Diffraction Analysis ◽  
Active Site ◽  
Quaternary Structure ◽  
Cross Reactivity ◽  
Polyclonal Antiserum ◽  
Wild Type ◽  
X Ray Diffraction ◽  
Wild Type Enzyme ◽  
X Ray

Wild-type and site-specific mutants C166S and C166A (Cys-166 → Ser and Cys-166 → Ala respectively) of the amidase (acylamide amidohydrolase, EC 3.5.1.4) from Pseudomonas aeruginosa were expressed in Escherichia coli by using the vector pKK223-3. Both mutant proteins were catalytically inactive but showed complete cross-reactivity with polyclonal antiserum raised against the wild-type enzyme, as well as CD spectra identical with that of the wild-type enzyme, which were indicative of correct folding. Cys-166 is therefore implicated as the active-site nucleophile. Titration of free thiol groups with 5,5ʹ-dithiobis-(2-nitrobenzoic acid) indicated that Cys-166 is not a rapidly reacting residue. Crystals of both wild-type and C166S amidase grew with identical, rhombohedral morphology; X-ray diffraction analysis established the unit cell dimensions (a = b = c = 84 Å; α = β = γ = 75 °) and space group (R3 or R32). These results imply a quaternary structure of six subunits, with most probably 32 symmetry; the existence of a hexameric structure was supported by molecular mass determinations based on gel filtration and electrophoretic mobility.

scholarly journals Evidence that cysteine-166 is the active-site nucleophile of Pseudomonas aeruginosa amidase: crystallization and preliminary X-ray diffraction analysis of the enzyme

1999 ◽  
Vol 340 (3) ◽  
pp. 711 ◽  
Author(s):  
Sebastien FARNAUD ◽  
Renée TATA ◽  
Maninder K. SOHI ◽  
Tommy WAN ◽  
Paul R. BROWN ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Diffraction Analysis ◽  
Active Site ◽  
X Ray Diffraction ◽  
X Ray

scholarly journals The Structure of the Dizinc Subclass B2 Metallo-β-Lactamase CphA Reveals that the Second Inhibitory Zinc Ion Binds in the Histidine Site

2009 ◽  
Vol 53 (10) ◽  
pp. 4464-4471 ◽  
Author(s):  
Carine Bebrone ◽  
Heinrich Delbrück ◽  
Michaël B. Kupper ◽  
Philipp Schlömer ◽  
Charlotte Willmann ◽  
...  
Keyword(s):  
Active Site ◽  
Aeromonas Hydrophila ◽  
Zinc Ion ◽  
Amide Bond ◽  
Zinc Ions ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
X Ray ◽  
Metal Ligands ◽  
Class B

ABSTRACT Bacteria can defend themselves against β-lactam antibiotics through the expression of class B β-lactamases, which cleave the β-lactam amide bond and render the molecule harmless. There are three subclasses of class B β-lactamases (B1, B2, and B3), all of which require Zn2+ for activity and can bind either one or two zinc ions. Whereas the B1 and B3 metallo-β-lactamases are most active as dizinc enzymes, subclass B2 enzymes, such as Aeromonas hydrophila CphA, are inhibited by the binding of a second zinc ion. We crystallized A. hydrophila CphA in order to determine the binding site of the inhibitory zinc ion. X-ray data from zinc-saturated crystals allowed us to solve the crystal structures of the dizinc forms of the wild-type enzyme and N220G mutant. The first zinc ion binds in the cysteine site, as previously determined for the monozinc form of the enzyme. The second zinc ion occupies a slightly modified histidine site, where the conserved His118 and His196 residues act as metal ligands. This atypical coordination sphere probably explains the rather high dissociation constant for the second zinc ion compared to those observed with enzymes of subclasses B1 and B3. Inhibition by the second zinc ion results from immobilization of the catalytically important His118 and His196 residues, as well as the folding of the Gly232-Asn233 loop into a position that covers the active site.

Structural and functional insights into an archaealL-asparaginase obtained through the linker-less assembly of constituent domains

2014 ◽  
Vol 70 (12) ◽  
pp. 3187-3197 ◽  
Author(s):  
Rachana Tomar ◽  
Pankaj Sharma ◽  
Ankit Srivastava ◽  
Saurabh Bansal ◽  
Ashish ◽  
...  
Keyword(s):  
Active Site ◽  
Elevated Temperatures ◽  
Quaternary Structure ◽  
Pyrococcus Furiosus ◽  
Multidomain Proteins ◽  
Wild Type ◽  
X Ray ◽  
Data Comparison ◽  
And Function ◽  
Tetrameric Form

Covalent linkers bridging the domains of multidomain proteins are considered to be crucial for assembly and function. In this report, an exception in which the linker of a two-domain dimeric L-asparaginase fromPyrococcus furiosus(PfA) was found to be dispensable is presented. Domains of this enzyme assembled without the linker into a conjoined tetrameric form that exhibited higher activity than the parent enzyme. The global shape and quaternary structure of the conjoined PfA were also similar to the wild-type PfA, as observed by their solution scattering profiles and X-ray crystallographic data. Comparison of the crystal structures of substrate-bound and unbound enzymes revealed an altogether new active-site composition and mechanism of action. Thus, conjoined PfA is presented as a unique enzyme obtained through noncovalent, linker-less assembly of constituent domains that is stable enough to function efficiently at elevated temperatures.

A New Germacranolide and a New Ceramide from Salvia nubicola (Lamiaceae)

2007 ◽  
Vol 62 (10) ◽  
pp. 1333-1338 ◽  
Author(s):  
Muhammad S. Ali ◽  
Syed A. Ibrahima ◽  
Shakeel Ahmed ◽  
Emil Lobkovsky
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Growth Inhibition ◽  
Single Crystal ◽  
Diffraction Analysis ◽  
Lemna Minor ◽  
Spectroscopic Techniques ◽  
X Ray Diffraction ◽  
Strong Growth ◽  
X Ray ◽  
Soluble Part

The methanol-soluble part of Salvia nubicola has yielded a new germacranolide and a new ceramide named nubtrienolide and nubenamide, respectively. Structures of both the isolated constituents were elucidated with the aid of spectroscopic techniques; the nubtrienolide structure was further confirmed via single crystal X-ray diffraction analysis. Among both the metabolites, nubtrienolide showed strong growth inhibition against Pseudomonas aeruginosa while during phytotoxicity testing, the same compound promoted the growth of Lemna minor L. instead to inhibit it.

scholarly journals Cleavage of nicotinamide adenine dinucleotide by the ribosome-inactivating protein fromMomordica charantia

2015 ◽  
Vol 71 (9) ◽  
pp. 1152-1155 ◽  
Author(s):  
M. Vinkovic ◽  
G. Dunn ◽  
G. E. Wood ◽  
J. Husain ◽  
S. P. Wood ◽  
...  
Keyword(s):  
Diffraction Analysis ◽  
Active Site ◽  
Nicotinamide Adenine Dinucleotide ◽  
Membered Ring ◽  
Enzyme Mechanism ◽  
X Ray Diffraction ◽  
Ribosome Inactivating Protein ◽  
X Ray ◽  
Enzyme Active Site

The interaction of momordin, a type 1 ribosome-inactivating protein fromMomordica charantia, with NADP+and NADPH has been investigated by X-ray diffraction analysis of complexes generated by co-crystallization and crystal soaking. It is known that the proteins of this family readily cleave the adenine–ribose bond of adenosine and related nucleotides in the crystal, leaving the product, adenine, bound to the enzyme active site. Surprisingly, the nicotinamide–ribose bond of oxidized NADP+is cleaved, leaving nicotinamide bound in the active site in the same position but in a slightly different orientation to that of the five-membered ring of adenine. No binding or cleavage of NADPH was observed at pH 7.4 in these experiments. These observations are in accord with current views of the enzyme mechanism and may contribute to ongoing searches for effective inhibitors.

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