Role of the Active Site Gate of Glycogen Phosphorylase in Allosteric Inhibition and Substrate Binding

AbstractCytochrome P450 2J2 (CYP2J2) is responsible for the epoxidation of endogenous arachidonic acid, and is involved in the metabolism of exogenous drugs. To date, no crystal structure of CYP2J2 is available, and the proposed structural basis for the substrate recognition and specificity in CYP2J2 varies with the structural models developed using different computational protocols. In this study, we developed a new structural model of CYP2J2, and explored its sensitivity to substrate binding by molecular dynamics simulations of the interactions with chemically similar fluorescent probes. Our results showed that the induced-fit binding of these probes led to the preferred active poses ready for the catalysis by CYP2J2. Divergent conformational dynamics of CYP2J2 due to the binding of each probe were observed. However, a stable hydrophobic clamp composed of residues I127, F310, A311, V380, and I487 was identified to restrict any substrate access to the active site of CYP2J2. Molecular docking of a series of compounds including amiodarone, astemizole, danazol, ebastine, ketoconazole, terfenadine, terfenadone, and arachidonic acid to CYP2J2 confirmed the role of those residues in determining substrate binding and specificity of CYP2J2. In addition to the flexibility of CYP2J2, the present work also identified other factors such as electrostatic potential in the vicinity of the active site, and substrate strain energy and property that have implications for the interpretation of CYP2J2 metabolism.

Download Full-text

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

Applied and Environmental Microbiology ◽

10.1128/aem.00687-11 ◽

2011 ◽

Vol 77 (16) ◽

pp. 5730-5738 ◽

Cited By ~ 36

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.

Download Full-text

Tissue transglutaminase acylation: Proposed role of conserved active site Tyr and Trp residues revealed by molecular modeling of peptide substrate binding

Protein Science ◽

10.1110/ps.03433304 ◽

2004 ◽

Vol 13 (4) ◽

pp. 979-991 ◽

Cited By ~ 31

Author(s):

R. A. Chica

Keyword(s):

Molecular Modeling ◽

Active Site ◽

Tissue Transglutaminase ◽

Substrate Binding ◽

Peptide Substrate ◽

Peptide Substrate Binding

Download Full-text

Trp–His covalent adduct in bilirubin oxidase is crucial for effective bilirubin binding but has a minor role in electron transfer

Scientific Reports ◽

10.1038/s41598-019-50105-3 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Tomáš Kovaľ ◽

Leona Švecová ◽

Lars H. Østergaard ◽

Tereza Skalova ◽

Jarmila Dušková ◽

...

Keyword(s):

Electron Transfer ◽

Active Site ◽

Substrate Binding ◽

Minor Role ◽

Post Translational Modification ◽

Unique Type ◽

Bilirubin Oxidase ◽

Bilirubin Binding ◽

A Minor

Abstract Unlike any protein studied so far, the active site of bilirubin oxidase from Myrothecium verrucaria contains a unique type of covalent link between tryptophan and histidine side chains. The role of this post-translational modification in substrate binding and oxidation is not sufficiently understood. Our structural and mutational studies provide evidence that this Trp396–His398 adduct modifies T1 copper coordination and is an important part of the substrate binding and oxidation site. The presence of the adduct is crucial for oxidation of substituted phenols and it substantially influences the rate of oxidation of bilirubin. Additionally, we bring the first structure of bilirubin oxidase in complex with one of its products, ferricyanide ion, interacting with the modified tryptophan side chain, Arg356 and the active site-forming loop 393-398. The results imply that structurally and chemically distinct types of substrates, including bilirubin, utilize the Trp–His adduct mainly for binding and to a smaller extent for electron transfer.

Download Full-text