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.

The role of Glu196 in the environment around the substrate binding site of leucine aminopeptidase fromStreptomyces griseus

FEBS Letters ◽  
2006 ◽  
Vol 580 (3) ◽  
pp. 912-917 ◽  
Author(s):  
Jiro Arima ◽  
Yoshiko Uesugi ◽  
Misugi Uraji ◽  
Masaki Iwabuchi ◽  
Tadashi Hatanaka
Keyword(s):  
Binding Site ◽  
Leucine Aminopeptidase ◽  
Substrate Binding ◽  
Substrate Binding Site

scholarly journals The Role of the Conserved Glycines of ATP-binding Cassette Signature Motifs of MRP1 in the Communication between the Substrate-binding Site and the Catalytic Centers

2004 ◽  
Vol 279 (40) ◽  
pp. 41670-41678 ◽  
Author(s):  
Zsófia Szentpétery ◽  
András Kern ◽  
Károly Liliom ◽  
Balázs Sarkadi ◽  
András Váradi ◽  
...  
Keyword(s):  
Binding Site ◽  
Substrate Binding ◽  
Atp Binding ◽  
Substrate Binding Site ◽  
Atp Binding Cassette

scholarly journals Kinetic, Structural, and Mutational Analysis of Acyl-CoA Carboxylase From Thermobifida fusca YX

2021 ◽  
Vol 7 ◽  
Author(s):  
Kiran-Kumar Shivaiah ◽  
Bryon Upton ◽  
Basil J. Nikolau
Keyword(s):  
Substrate Specificity ◽  
Quaternary Structure ◽  
Mutational Analysis ◽  
Streptomyces Coelicolor ◽  
Thermobifida Fusca ◽  
Substrate Selectivity ◽  
Acetyl Coa ◽  
Catalytic Subunits ◽  
Chain Acyl

Acyl-CoA carboxylases (AcCCase) are biotin-dependent enzymes that are capable of carboxylating more than one short chain acyl-CoA substrate. We have conducted structural and kinetic analyses of such an AcCCase from Thermobifida fusca YX, which exhibits promiscuity in carboxylating acetyl-CoA, propionyl-CoA, and butyryl-CoA. The enzyme consists of two catalytic subunits (TfAcCCA and TfAcCCB) and a non-catalytic subunit, TfAcCCE, and is organized in quaternary structure with a A6B6E6 stoichiometry. Moreover, this holoenzyme structure appears to be primarily assembled from two A3 and a B6E6 subcomplexes. The role of the TfAcCCE subunit is to facilitate the assembly of the holoenzyme complex, and thereby activate catalysis. Based on prior studies of an AcCCase from Streptomyces coelicolor, we explored whether a conserved Asp residue in the TfAcCCB subunit may have a role in determining the substrate selectivity of these types of enzymes. Mutating this D427 residue resulted in alterations in the substrate specificity of the TfAcCCase, increasing proficiency for carboxylating acetyl-CoA, while decreasing carboxylation proficiency with propionyl-CoA and butyryl-CoA. Collectively these results suggest that residue D427 of AcCCB subunits is an important, but not sole determinant of the substrate specificity of AcCCase enzymes.

scholarly journals Molecular determinant of substrate binding and specificity of cytochrome P450 2J2

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Liang Xu ◽  
Liao Y. Chen
Keyword(s):  
Arachidonic Acid ◽  
Cytochrome P450 ◽  
Active Site ◽  
Structural Model ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Structural Basis ◽  
Molecular Determinant ◽  
Dynamics Simulations

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.

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