Crystal structures and functional studies clarify substrate selectivity and catalytic residues for the unique orphan enzyme N-acetyl-D-mannosamine dehydrogenase

2014 ◽  
Vol 462 (3) ◽  
pp. 499-511 ◽  
Author(s):  
Agustín Sola-Carvajal ◽  
Fernando Gil-Ortiz ◽  
Francisco García-Carmona ◽  
Vicente Rubio ◽  
Álvaro Sánchez-Ferrer
Keyword(s):  
Substrate Specificity ◽  
Crystal Structures ◽  
Functional Site ◽  
Site Directed Mutagenesis ◽  
Short Chain ◽  
Directed Mutagenesis ◽  
Substrate Selectivity ◽  
Functional Studies ◽  
Short Chain Dehydrogenase ◽  
Orphan Enzyme

Functional site-directed mutagenesis and crystallographic studies with N-acetyl-D-mannosamine dehydrogenase reveal a homotetramer, a short-chain dehydrogenase/reductase subunit fold and a highly developed C-terminal tail interlinking subunit. The structures of the complexes explain substrate specificity. The four residues forming the catalytic tetrad are identified.

scholarly journals Structural basis for substrate binding and specificity of a sodium–alanine symporter AgcS

2019 ◽  
Vol 116 (6) ◽  
pp. 2086-2090 ◽  
Author(s):  
Jinming Ma ◽  
Hsiang-Ting Lei ◽  
Francis E. Reyes ◽  
Silvia Sanchez-Martinez ◽  
Maen F. Sarhan ◽  
...  
Keyword(s):  
Organic Molecules ◽  
Substrate Binding ◽  
Structural Features ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Directed Mutagenesis ◽  
Substrate Selectivity ◽  
Methanococcus Maripaludis ◽  
Functional Studies ◽  
Key Residues

The amino acid, polyamine, and organocation (APC) superfamily is the second largest superfamily of membrane proteins forming secondary transporters that move a range of organic molecules across the cell membrane. Each transporter in the APC superfamily is specific for a unique subset of substrates, even if they possess a similar structural fold. The mechanism of substrate selectivity remains, by and large, elusive. Here, we report two crystal structures of an APC member fromMethanococcus maripaludis, the alanine or glycine:cation symporter (AgcS), withl- ord-alanine bound. Structural analysis combined with site-directed mutagenesis and functional studies inform on substrate binding, specificity, and modulation of the AgcS family and reveal key structural features that allow this transporter to accommodate glycine and alanine while excluding all other amino acids. Mutation of key residues in the substrate binding site expand the selectivity to include valine and leucine. These studies provide initial insights into substrate selectivity in AgcS symporters.

scholarly journals Study of substrate specificity of human aromatase by site directed mutagenesis

2002 ◽  
Vol 269 (5) ◽  
pp. 1393-1405 ◽  
Author(s):  
P. Auvray ◽  
C. Nativelle ◽  
R. Bureau ◽  
P. Dallemagne ◽  
G.-E. Séralini ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Human Aromatase

scholarly journals Versatile Fungal Polyphenol Oxidase with Chlorophenol Bioremediation Potential: Characterization and Protein Engineering

2018 ◽  
Vol 84 (23) ◽  
Author(s):  
Efstratios Nikolaivits ◽  
Maria Dimarogona ◽  
Ioanna Karagiannaki ◽  
Angelina Chalima ◽  
Ayelet Fishman ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Fruits And Vegetables ◽  
Homology Model ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Gene Encoding ◽  
Content Type ◽  
Polyphenol Oxidases ◽  
Increased Activity ◽  
Work Knowledge

ABSTRACTPolyphenol oxidases (PPOs) have been mostly associated with the undesirable postharvest browning in fruits and vegetables and have implications in human melanogenesis. Nonetheless, they are considered useful biocatalysts in the food, pharmaceutical, and cosmetic industries. The aim of the present work was to characterize a novel PPO and explore its potential as a bioremediation agent. A gene encoding an extracellular tyrosinase-like enzyme was amplified from the genome ofThermothelomyces thermophilaand expressed inPichia pastoris. The recombinant enzyme (TtPPO) was purified and biochemically characterized. Its production reached 40 mg/liter, and it appeared to be a glycosylated and N-terminally processed protein.TtPPO showed broad substrate specificity, as it could oxidize 28/30 compounds tested, including polyphenols, substituted phenols, catechols, and methoxyphenols. Its optimum temperature was 65°C, with a half-life of 18.3 h at 50°C, while its optimum pH was 7.5. The homology model ofTtPPO was constructed, and site-directed mutagenesis was performed in order to increase its activity on mono- and dichlorophenols (di-CPs). The G292N/Y296V variant ofTtPPO 5.3-fold increased activity on 3,5-dichlorophenol (3,5-diCP) compared to the wild type.IMPORTANCEA novel fungal PPO was heterologously expressed and biochemically characterized. Construction of single and double mutants led to the generation of variants with altered specificity against CPs. Through this work, knowledge is gained regarding the effect of mutations on the substrate specificity of PPOs. This work also demonstrates that more potent biocatalysts for the bioremediation of harmful CPs can be developed by applying site-directed mutagenesis.

scholarly journals Domain sliding of two Staphylococcus aureus N-acetylglucosaminidases enables their substrate-binding prior to its catalysis

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Sara Pintar ◽  
Jure Borišek ◽  
Aleksandra Usenik ◽  
Andrej Perdih ◽  
Dušan Turk
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Drug Targets ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Human Pathogen ◽  
Active Site Cleft ◽  
Novel Drug ◽  
Novel Drug Targets

AbstractTo achieve productive binding, enzymes and substrates must align their geometries to complement each other along an entire substrate binding site, which may require enzyme flexibility. In pursuit of novel drug targets for the human pathogen S. aureus, we studied peptidoglycan N-acetylglucosaminidases, whose structures are composed of two domains forming a V-shaped active site cleft. Combined insights from crystal structures supported by site-directed mutagenesis, modeling, and molecular dynamics enabled us to elucidate the substrate binding mechanism of SagB and AtlA-gl. This mechanism requires domain sliding from the open form observed in their crystal structures, leading to polysaccharide substrate binding in the closed form, which can enzymatically process the bound substrate. We suggest that these two hydrolases must exhibit unusual extents of flexibility to cleave the rigid structure of a bacterial cell wall.

scholarly journals Enhanced Activity and Substrate Specificity by Site‐Directed Mutagenesis for the P450 119 Peroxygenase Catalyzed Sulfoxidation of Thioanisole

ChemistryOpen ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 1076-1083
Author(s):  
Xiaoyao Wei ◽  
Chun Zhang ◽  
Xiaowei Gao ◽  
Yanping Gao ◽  
Ya Yang ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Enhanced Activity
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