Biochemical and structural characterization of quinoprotein aldose sugar dehydrogenase from Thermus thermophilus HJ6: Mutational analysis of Tyr156 in the substrate-binding site

2016 ◽  
Vol 608 ◽  
pp. 20-26 ◽  
Author(s):  
Han-Woo Kim ◽  
Ji-Yeon Wang ◽  
Ji-Yeon Lee ◽  
Ae-Kyung Park ◽  
Hyun Park ◽  
...  
Keyword(s):  
Binding Site ◽  
Structural Characterization ◽  
Mutational Analysis ◽  
Substrate Binding ◽  
Thermus Thermophilus ◽  
Substrate Binding Site

scholarly journals Molecular Characterization of the Principal Substrate Binding Site of the Ubiquitous Folding Catalyst Protein Disulfide Isomerase

2003 ◽  
Vol 279 (11) ◽  
pp. 10374-10381 ◽  
Author(s):  
Annamari Pirneskoski ◽  
Peter Klappa ◽  
Mario Lobell ◽  
Richard A. Williamson ◽  
Lee Byrne ◽  
...  
Keyword(s):  
Binding Site ◽  
Molecular Characterization ◽  
Protein Disulfide Isomerase ◽  
Substrate Binding ◽  
Disulfide Isomerase ◽  
Substrate Binding Site ◽  
Protein Disulfide

NMR solution structure and characterization of substrate binding site of the PPIase domain of PrsA protein fromBacillus subtilis

FEBS Letters ◽  
2006 ◽  
Vol 580 (7) ◽  
pp. 1822-1826 ◽  
Author(s):  
Helena Tossavainen ◽  
Perttu Permi ◽  
Susanna L. Purhonen ◽  
Matti Sarvas ◽  
Ilkka Kilpeläinen ◽  
...  
Keyword(s):  
Binding Site ◽  
Solution Structure ◽  
Substrate Binding ◽  
Nmr Solution Structure ◽  
Substrate Binding Site ◽  
Ppiase Domain

scholarly journals Mapping the Substrate Binding Site of Phenylacetone Monooxygenase from Thermobifida fusca by Mutational Analysis

2011 ◽  
Vol 77 (16) ◽  
pp. 5730-5738 ◽  
Author(s):  
Hanna M. Dudek ◽  
Gonzalo de Gonzalo ◽  
Daniel E. Torres Pazmiño ◽  
Piotr Stępniak ◽  
Lucjan S. Wyrwicz ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Binding Site ◽  
Active Site ◽  
Structural Model ◽  
Mutational Analysis ◽  
Substrate Binding ◽  
Thermobifida Fusca ◽  
Content Type ◽  
Substrate Binding Site

ABSTRACTBaeyer-Villiger monooxygenases catalyze oxidations that are of interest for biocatalytic applications. Among these enzymes, phenylacetone monooxygenase (PAMO) fromThermobifida fuscais the only protein showing remarkable stability. While related enzymes often present a broad substrate scope, PAMO accepts only a limited number of substrates. Due to the absence of a substrate in the elucidated crystal structure of PAMO, the substrate binding site of this protein has not yet been defined. In this study, a structural model of cyclopentanone monooxygenase, which acts on a broad range of compounds, has been prepared and compared with the structure of PAMO. This revealed 15 amino acid positions in the active site of PAMO that may account for its relatively narrow substrate specificity. We designed and analyzed 30 single and multiple mutants in order to verify the role of these positions. Extensive substrate screening revealed several mutants that displayed increased activity and altered regio- or enantioselectivity in Baeyer-Villiger reactions and sulfoxidations. Further substrate profiling resulted in the identification of mutants with improved catalytic properties toward synthetically attractive compounds. Moreover, the thermostability of the mutants was not compromised in comparison to that of the wild-type enzyme. Our data demonstrate that the positions identified within the active site of PAMO, namely, V54, I67, Q152, and A435, contribute to the substrate specificity of this enzyme. These findings will aid in more dedicated and effective redesign of PAMO and related monooxygenases toward an expanded substrate scope.

scholarly journals Mutational analysis of the RecA protein L1 region identifies this area as a probable part of the co‐protease substrate binding site

1997 ◽  
Vol 25 (5) ◽  
pp. 967-978 ◽  
Author(s):  
Horacio G. Nastri ◽  
Angelina Guzzo ◽  
Craig S. Lange ◽  
Graham C. Walker ◽  
Kendall L. Knight
Keyword(s):  
Binding Site ◽  
Mutational Analysis ◽  
Substrate Binding ◽  
Reca Protein ◽  
Substrate Binding Site ◽  
Protease Substrate

scholarly journals Crystal Structure of Tetrameric Homoisocitrate Dehydrogenase from an Extreme Thermophile, Thermus thermophilus: Involvement of Hydrophobic Dimer-Dimer Interaction in Extremely High Thermotolerance

2005 ◽  
Vol 187 (19) ◽  
pp. 6779-6788 ◽  
Author(s):  
Junichi Miyazaki ◽  
Kuniko Asada ◽  
Shinya Fushinobu ◽  
Tomohisa Kuzuyama ◽  
Makoto Nishiyama
Keyword(s):  
Crystal Structure ◽  
Binding Site ◽  
Thermal Inactivation ◽  
Hydrophobic Interactions ◽  
Substrate Binding ◽  
Thermus Thermophilus ◽  
Lysine Biosynthesis ◽  
Side Chain ◽  
Substrate Binding Site ◽  
Tetramer Formation

ABSTRACT The crystal structure of homoisocitrate dehydrogenase involved in lysine biosynthesis from Thermus thermophilus (TtHICDH) was determined at 1.85-Å resolution. Arg85, which was shown to be a determinant for substrate specificity in our previous study, is positioned close to the putative substrate binding site and interacts with Glu122. Glu122 is highly conserved in the equivalent position in the primary sequence of ICDH and archaeal 3-isopropylmalate dehydrogenase (IPMDH) but interacts with main- and side-chain atoms in the same domain in those paralogs. In addition, a conserved Tyr residue (Tyr125 in TtHICDH) which extends its side chain toward a substrate and thus has a catalytic function in the related β-decarboxylating dehydrogenases, is flipped out of the substrate-binding site. These results suggest the possibility that the conformation of the region containing Glu122-Tyr125 is changed upon substrate binding in TtHICDH. The crystal structure of TtHICDH also reveals that the arm region is involved in tetramer formation via hydrophobic interactions and might be responsible for the high thermotolerance. Mutation of Val135, located in the dimer-dimer interface and involved in the hydrophobic interaction, to Met alters the enzyme to a dimer (probably due to steric perturbation) and markedly decreases the thermal inactivation temperature. Both the crystal structure and the mutation analysis indicate that tetramer formation is involved in the extremely high thermotolerance of TtHICDH.

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