scholarly journals Kinetic and crystallographic analyses of the catalytic domain of chitinase from Pyrococcus furiosus- the role of conserved residues in the active site

FEBS Journal ◽  
2010 ◽  
Vol 277 (12) ◽  
pp. 2683-2695 ◽  
Author(s):  
Hiroaki Tsuji ◽  
Shigenori Nishimura ◽  
Takashi Inui ◽  
Yuji Kado ◽  
Kazuhiko Ishikawa ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Domain ◽  
Pyrococcus Furiosus ◽  
Conserved Residues

scholarly journals GlcNAcstatins are nanomolar inhibitors of human O-GlcNAcase inducing cellular hyper-O-GlcNAcylation

2009 ◽  
Vol 420 (2) ◽  
pp. 221-227 ◽  
Author(s):  
Helge C. Dorfmueller ◽  
Vladimir S. Borodkin ◽  
Marianne Schimpl ◽  
Daan M. F. van Aalten
Keyword(s):  
Active Site ◽  
Cell Biology ◽  
Rational Design ◽  
Catalytic Mechanism ◽  
Conserved Residues ◽  
Cellular Processes ◽  
Acetyl Group ◽  
Nanomolar Range ◽  
Insight Into

O-GlcNAcylation is an essential, dynamic and inducible post-translational glycosylation of cytosolic proteins in metazoa and can show interplay with protein phosphorylation. Inhibition of OGA (O-GlcNAcase), the enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, is a useful strategy to probe the role of this modification in a range of cellular processes. In the present study, we report the rational design and evaluation of GlcNAcstatins, a family of potent, competitive and selective inhibitors of human OGA. Kinetic experiments with recombinant human OGA reveal that the GlcNAcstatins are the most potent human OGA inhibitors reported to date, inhibiting the enzyme in the sub-nanomolar to nanomolar range. Modification of the GlcNAcstatin N-acetyl group leads to up to 160-fold selectivity against the human lysosomal hexosaminidases which employ a similar substrate-assisted catalytic mechanism. Mutagenesis studies in a bacterial OGA, guided by the structure of a GlcNAcstatin complex, provides insight into the role of conserved residues in the human OGA active site. GlcNAcstatins are cell-permeant and, at low nanomolar concentrations, effectively modulate intracellular O-GlcNAc levels through inhibition of OGA, in a range of human cell lines. Thus these compounds are potent selective tools to study the cell biology of O-GlcNAc.

scholarly journals Substrate-modulated Cytochrome P450 17A1 and Cytochrome b5 Interactions Revealed by NMR

2013 ◽  
Vol 288 (23) ◽  
pp. 17008-17018 ◽  
Author(s):  
D. Fernando Estrada ◽  
Jennifer S. Laurence ◽  
Emily E. Scott
Keyword(s):  
Cytochrome P450 ◽  
Binding Site ◽  
Active Site ◽  
Catalytic Domain ◽  
Cytochrome B5 ◽  
Structural Basis ◽  
Reaction Conditions ◽  
Reversible Binding ◽  
Important Interaction

The membrane heme protein cytochrome b5 (b5) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Here the interactions between the soluble domain of microsomal b5 and the catalytic domain of the bifunctional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b5, and an androgen-forming lyase reaction that is facilitated 10-fold by b5. NMR chemical shift mapping of b5 titrations with CYP17A1 indicates that the interaction occurs in an intermediate exchange regime and identifies charged surface residues involved in the protein/protein interface. The role of these residues is confirmed by disruption of the complex upon mutagenesis of either the anionic b5 residues (Glu-48 or Glu-49) or the corresponding cationic CYP17A1 residues (Arg-347, Arg-358, or Arg-449). Cytochrome b5 binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase. To probe the differential effects of b5 on the two CYP17A1-mediated reactions and, thus, communication between the superficial b5 binding site and the buried CYP17A1 active site, CYP17A1/b5 complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b5 interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17α-hydroxypregnenolone is in the active site. These findings form the basis for a clearer understanding of this important interaction by directly measuring the reversible binding of the two proteins, providing evidence of communication between the CYP17A1 active site and the superficial proximal b5 binding site.

Active site characterization and activity of the human aspartyl (asparaginyl) β-hydroxylase

Metallomics ◽  
2021 ◽  
Vol 13 (10) ◽  
Author(s):  
Jenna M Greve ◽  
Andrew M Pinkham ◽  
Zechariah Thompson ◽  
J A Cowan
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Catalytic Domain ◽  
Negative Impact ◽  
Enzyme Stability ◽  
Titration Calorimetry ◽  
Cellular Biochemistry ◽  
Biophysical Approach

Abstract Human aspartyl/asparaginyl beta-hydroxylase (HAAH) is a member of the superfamily of nonheme Fe2+/α-ketoglutarate (αKG) dependent oxygenase enzymes with a noncanonical active site. HAAH hydroxylates epidermal growth factor (EGF) like domains to form the β-hydroxylated product from substrate asparagine or aspartic acid and has been suggested to have a negative impact in a variety of cancers. In addition to iron, HAAH also binds divalent calcium, although the role of the latter is not understood. Herein, the metal binding chemistry and influence on enzyme stability and activity have been evaluated by a combined biochemical and biophysical approach. Metal binding parameters for the HAAH active site were determined by use of isothermal titration calorimetry, demonstrating a high-affinity regulatory binding site for Ca2+ in the catalytic domain in addition to the catalytic Fe2+ cofactor. We have analyzed various active site derivatives, utilizing LC-MS and a new HPLC technique to determine the role of metal binding and the second coordination sphere in enzyme activity, discovering a previously unreported residue as vital for HAAH turnover. This analysis of the in vitro biochemical function of HAAH furthers the understanding of its importance to cellular biochemistry and metabolic pathways.

scholarly journals Mutational and computational analysis of the role of conserved residues in the active site of a family 18 chitinase

2004 ◽  
Vol 271 (2) ◽  
pp. 253-262 ◽  
Author(s):  
Bjornar Synstad ◽  
Sigrid Gaseidnes ◽  
Daan M. F. van Aalten ◽  
Gert Vriend ◽  
Jens E Nielsen ◽  
...  
Keyword(s):  
Active Site ◽  
Computational Analysis ◽  
Conserved Residues

scholarly journals Dual Active Site in the Endolytic Transglycosylase gp144 of Bacteriophage phiKZ

Acta Naturae ◽  
2017 ◽  
Vol 9 (1) ◽  
pp. 81-87 ◽  
Author(s):  
O. V. Chertkov ◽  
G. A. Armeev ◽  
I. V. Uporov ◽  
S. A. Legotsky ◽  
N. N. Sykilinda ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Catalytic Domain ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Lytic Transglycosylases ◽  
Bacteriophage Infection ◽  
Surface Charge Distribution ◽  
Modular Composition

Lytic transglycosylases are abundant peptidoglycan lysing enzymes that degrade the heteropolymers of bacterial cell walls in metabolic processes or in the course of a bacteriophage infection. The conventional catalytic mechanism of transglycosylases involves only the Glu or Asp residue. Endolysin gp144 of Pseudomonas aeruginosa bacteriophage phiKZ belongs to the family of Gram-negative transglycosylases with a modular composition and C-terminal location of the catalytic domain. Glu115 of gp144 performs the predicted role of a catalytic residue. However, replacement of this residue does not completely eliminate the activity of the mutant protein. Site-directed mutagenesis has revealed the participation of Tyr197 in the catalytic mechanism, as well as the presence of a second active site involving Glu178 and Tyr147. The existence of the dual active site was supported by computer modeling and monitoring of the molecular dynamics of the changes in the conformation and surface charge distribution as a consequence of point mutations.

scholarly journals Crystal Structure of the Extended-Spectrum β-Lactamase PER-2 and Insights into the Role of Specific Residues in the Interaction with β-Lactams and β-Lactamase Inhibitors

2014 ◽  
Vol 58 (10) ◽  
pp. 5994-6002 ◽  
Author(s):  
Melina Ruggiero ◽  
Frédéric Kerff ◽  
Raphaël Herman ◽  
Frédéric Sapunaric ◽  
Moreno Galleni ◽  
...  
Keyword(s):  
Active Site ◽  
Amino Acid Identity ◽  
Conserved Residues ◽  
Class A ◽  
Extended Spectrum ◽  
Active Site Cavity ◽  
Bond Network ◽  
Hydrolysis Of ◽  
Structure And Activity

ABSTRACTPER-2 belongs to a small (7 members to date) group of extended-spectrum β-lactamases. It has 88% amino acid identity with PER-1 and both display high catalytic efficiencies toward most β-lactams. In this study, we determined the X-ray structure of PER-2 at 2.20 Å and evaluated the possible role of several residues in the structure and activity toward β-lactams and mechanism-based inhibitors. PER-2 is defined by the presence of a singulartransbond between residues 166 to 167, which generates an inverted Ω loop, an expanded fold of this domain that results in a wide active site cavity that allows for efficient hydrolysis of antibiotics like the oxyimino-cephalosporins, and a series of exclusive interactions between residues not frequently involved in the stabilization of the active site in other class A β-lactamases. PER β-lactamases might be included within a cluster of evolutionarily related enzymes harboring the conserved residues Asp136 and Asn179. Other signature residues that define these enzymes seem to be Gln69, Arg220, Thr237, and probably Arg/Lys240A (“A” indicates an insertion according to Ambler's scheme for residue numbering in PER β-lactamases), with structurally important roles in the stabilization of the active site and proper orientation of catalytic water molecules, among others. We propose, supported by simulated models of PER-2 in combination with different β-lactams, the presence of a hydrogen-bond network connecting Ser70-Gln69-water-Thr237-Arg220 that might be important for the proper activity and inhibition of the enzyme. Therefore, we expect that mutations occurring in these positions will have impacts on the overall hydrolytic behavior.

Sign in / Sign up

Export Citation Format

Share Document