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

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.

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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.

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Substrate selectivity of C-terminal sucrase isomaltase and maltase glucoamylase

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314091864 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C813-C813

Author(s):

Kyra Jones ◽

David Rose

Keyword(s):

Small Intestine ◽

Substrate Specificity ◽

Type Ii Diabetes ◽

Serum Insulin ◽

Substrate Selectivity ◽

Type Ii ◽

Significant Component ◽

Human Diet ◽

Catalytic Subunits ◽

Free Glucose

Carbohydrates make up a significant component of the human diet. One approach to controlling blood glucose and serum insulin levels in individuals with type II diabetes is inhibition of intestinal α-glucosidases and pancreatic α-amylases. Two intestinal α-glucosidases, sucrase isomaltase (SI) and maltase glucoamylase (MGAM), are responsible for the final step of starch hydrolysis in mammals in the small intestine: the release of free glucose. Each enzyme consists of two catalytic subunits: N-terminal sucrase isomaltase (ntSI) and C-terminal sucrase isomaltose (ctSI); and N-terminal maltase glucoamylase (ntMGAM) and C-terminal maltase glucoamylase (ctMGAM). Here, residues hypothesized to impact substrate specificity of ctSI and ctMGAM will be presented, enhancing our understanding of the functionality of these enzymatic subunits as well as their overlapping substrate specificity.

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The role of rice bran extract on acetyl CoA carboxylase in liver of rats fed a high-fat diet

Planta Medica ◽

10.1055/s-0031-1282786 ◽

2011 ◽

Vol 77 (12) ◽

Author(s):

C Charkhonpunya ◽

S Sireeratawong ◽

S Komindr ◽

N Lerdvuthisopon

Keyword(s):

Rice Bran ◽

High Fat Diet ◽

Acetyl Coa Carboxylase ◽

High Fat ◽

Acetyl Coa ◽

Rice Bran Extract

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Altering the Substrate Specificity of Acetyl-CoA Synthetase by Rational Mutagenesis of the Carboxylate Binding Pocket

ACS Synthetic Biology ◽

10.1021/acssynbio.9b00008 ◽

2019 ◽

Vol 8 (6) ◽

pp. 1325-1336 ◽

Cited By ~ 6

Author(s):

Naazneen Sofeo ◽

Jason H. Hart ◽

Brandon Butler ◽

David J. Oliver ◽

Marna D. Yandeau-Nelson ◽

...

Keyword(s):

Substrate Specificity ◽

Binding Pocket ◽

Acetyl Coa ◽

Rational Mutagenesis

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PRKAR2A deficiency protects mice from experimental colitis by increasing IFN-stimulated gene expression and modulating the intestinal microbiota

Mucosal Immunology ◽

10.1038/s41385-021-00426-2 ◽

2021 ◽

Author(s):

Lumin Wei ◽

Rongjing Zhang ◽

Jinzhao Zhang ◽

Juanjuan Li ◽

Deping Kong ◽

...

Keyword(s):

Experimental Colitis ◽

Regulatory Subunit ◽

Protective Effects ◽

Intestinal Epithelial ◽

Catalytic Subunits ◽

Regulatory Subunits ◽

Pka Regulatory Subunit ◽

Cross Fostering ◽

Specific Deletion

AbstractProtein kinase A (PKA) plays an important role in regulating inflammation via its catalytic subunits. Recently, PKA regulatory subunits have been reported to directly modulate some signaling pathways and alleviate inflammation. However, the role of PKA regulatory subunits in colonic inflammation remains unclear. Therefore, we conducted this study to investigate the role of the PKA regulatory subunit PRKAR2A in colitis. We observed that PRKAR2A deficiency protected mice from dextran sulfate sodium (DSS)-induced experimental colitis. Our experiments revealed that the intestinal epithelial cell-specific deletion of Prkar2a contributed to this protection. Mechanistically, the loss of PRKAR2A in Prkar2a−/− mice resulted in an increased IFN-stimulated gene (ISG) expression and altered gut microbiota. Inhibition of ISGs partially reversed the protective effects against DSS-induced colitis in Prkar2a−/− mice. Antibiotic treatment and cross-fostering experiments demonstrated that the protection against DSS-induced colitis in Prkar2a−/− mice was largely dependent on the gut microflora. Altogether, our work demonstrates a previously unidentified function of PRKAR2A in promoting DSS-induced colitis.

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Evidence for an essential role of long chain acyl-CoA synthetase in animal cell proliferation

Journal of Biological Chemistry ◽

10.1016/s0021-9258(20)64309-5 ◽

1991 ◽

Vol 266 (7) ◽

pp. 4214-4219

Author(s):

H Tomoda ◽

K Igarashi ◽

J C Cyong ◽

S Omura

Keyword(s):

Cell Proliferation ◽

Essential Role ◽

Animal Cell ◽

Chain Acyl ◽

Long Chain

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Analysis of Sequence Divergence in Mammalian ABCGs Predicts a Structural Network of Residues That Underlies Functional Divergence

International Journal of Molecular Sciences ◽

10.3390/ijms22063012 ◽

2021 ◽

Vol 22 (6) ◽

pp. 3012

Author(s):

James I. Mitchell-White ◽

Thomas Stockner ◽

Nicholas Holliday ◽

Stephen J. Briddon ◽

Ian D. Kerr

Keyword(s):

Substrate Specificity ◽

Functional Divergence ◽

3D Structure ◽

Sequence Divergence ◽

Conformational Flexibility ◽

Substrate Selectivity ◽

Multiple Sequence ◽

Atp Binding Cassette Transporters ◽

Structural Network ◽

Wide Substrate Specificity

The five members of the mammalian G subfamily of ATP-binding cassette transporters differ greatly in their substrate specificity. Four members of the subfamily are important in lipid transport and the wide substrate specificity of one of the members, ABCG2, is of significance due to its role in multidrug resistance. To explore the origin of substrate selectivity in members 1, 2, 4, 5 and 8 of this subfamily, we have analysed the differences in conservation between members in a multiple sequence alignment of ABCG sequences from mammals. Mapping sets of residues with similar patterns of conservation onto the resolved 3D structure of ABCG2 reveals possible explanations for differences in function, via a connected network of residues from the cytoplasmic to transmembrane domains. In ABCG2, this network of residues may confer extra conformational flexibility, enabling it to transport a wider array of substrates.

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