X-ray crystal structures of active site mutants of the vanadium-containing chloroperoxidase from the fungus Curvularia inaequalis

1999 ◽  
Vol 4 (2) ◽  
pp. 209-219 ◽  
Author(s):  
Sandra Macedo-Ribeiro ◽  
Wieger Hemrika ◽  
Rokus Renirie ◽  
Ron Wever ◽  
A. Messerschmidt
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
X Ray ◽  
Active Site Mutants

scholarly journals Structure based protein engineering to confer selectivity of guanine deaminase

2014 ◽  
Vol 70 (a1) ◽  
pp. C437-C437
Author(s):  
Aruna Bitra ◽  
Ruchi Anand
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Catalytic Efficiency ◽  
Structural Basis ◽  
Active Site Residues ◽  
X Ray ◽  
Secondary Activity ◽  
Guanine Deaminase ◽  
Differential Selectivity

Guanine deaminases (GDs) are important enzymes involved in both purine metabolism and nucleotide anabolism pathways. Here we present the molecular and catalytic mechanism of NE0047 and use the information obtained to engineer specific enzyme activities. NE0047 from Nitrosomonas europaea was found to be a high fidelity guanine deaminase (catalytic efficiency of 1.2 × 105 M–1 s–1). However; it exhibited secondary activity towards the structurally non-analogous triazine based compound ammeline. The X-ray structure of NE0047 in the presence of the substrate analogue 8-azaguanine help establish that the enzyme exists as a biological dimer and both the proper closure of the C-terminal loop and cross talk via the dimeric interface is crucial for conferring catalytic activity. It was further ascertained that the highly conserved active site residues Glu79 and Glu143 facilitate the deamination reaction by serving as proton shuttles. Moreover, to understand the structural basis of dual substrate specificity, X-ray structures of NE0047 in complex with a series of nucleobase analogs, nucleosides and substrate ammeline were determined. The crystal structures demonstrated that any substitutions in the parent substrates results in the rearrangement of the ligand in a catalytically unfavorable orientation and also impede the closure of catalytically important loop, thereby abrogating activity. However, ammeline was able to adopt a catalytically favorable orientation which, also allowed for proper loop closure. Based on the above knowledge of the crystal structures and the catalytic mechanism, the active site was subsequently engineered to fine-tune NE0047 activity. The mutated versions of the enzyme were designed so that they can function either exclusively as a GD or serve as specific ammeline deaminases. For example, mutations in the active site E143D and N66A confer the enzyme to be an unambiguous GD with no secondary activity towards ammeline. On the other hand, the N66Q mutant of NE0047 only deaminates ammeline. Additionally, a series of crystal structures of the mutant versions were solved that shed light on the structural basis of this differential selectivity.

scholarly journals Crystal Structures of E. coli Native MenH and Two Active Site Mutants

PLoS ONE ◽  
2013 ◽  
Vol 8 (4) ◽  
pp. e61325 ◽  
Author(s):  
Jodie M. Johnston ◽  
Ming Jiang ◽  
Zhihong Guo ◽  
Edward N. Baker
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
E Coli ◽  
Active Site Mutants

scholarly journals Chemical validation of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase using fragment linking and CRISPR interference

2020 ◽  
Author(s):  
Jamal El Bakali ◽  
Michal Blaszczyk ◽  
Joanna C. Evans ◽  
Jennifer A. Boland ◽  
William J. McCarthy ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Crystal Structures ◽  
Active Site ◽  
Drug Target ◽  
Potential Drug Target ◽  
Biosynthesis Pathway ◽  
Potential Drug ◽  
Antimicrobial Drugs ◽  
X Ray ◽  
Crispr Interference

AbstractThe coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify the first inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a KD < 20 μM and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug.Abstract Figure

Crystal structure of a pyrimidine dimer-specific excision repair enzyme from bacteriophage T4: Refinement at 1.45 Å and X-ray analysis of the three active site mutants

1995 ◽  
Vol 249 (2) ◽  
pp. 360-375 ◽  
Author(s):  
Kosuke Morikawa ◽  
Mariko Ariyoshi ◽  
Dmitry G. Vassylyev ◽  
Osamu Matsumoto ◽  
Katsuo Katayanagi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Excision Repair ◽  
Bacteriophage T4 ◽  
Pyrimidine Dimer ◽  
Repair Enzyme ◽  
X Ray ◽  
Active Site Mutants
Sign in / Sign up

Export Citation Format

Share Document