Allosteric Site at the Biotin Carboxylase Dimer Interface Mediates Activation and Inhibition in Staphylococcus aureus Pyruvate Carboxylase

Authors

Amanda J. Laseke, Marquette University
Trevor J. Boram, Purdue University
Nicholas O. Schneider, Marquette University
Jeremy R. Lohman, Purdue University
Martin St. Maurice, Marquette UniversityFollow

Document Type

Article

Publication Date

2023

Publisher

American Chemical Society

Source Publication

Biochemistry

Source ISSN

0006-2960

Original Item ID

DOI: 10.1021/acs.biochem.3c00280

Abstract

Allosteric regulation of the essential anaplerotic enzyme, pyruvate carboxylase (PC), is vital for metabolic homeostasis. PC catalyzes the bicarbonate- and ATP-dependent carboxylation of pyruvate to form oxaloacetate. Dysregulation of PC activity can impact glucose and redox metabolism, which contributes to the pathogenicity of many diseases. To maintain homeostasis, PC is allosterically activated by acetyl-CoA and allosterically inhibited by l-aspartate. In this study, we further characterize the molecular basis of allosteric regulation in Staphylococcus aureus PC (SaPC) using slowly/nonhydrolyzable dethia analogues of acetyl-CoA and site-directed mutagenesis of residues at the biotin carboxylase homodimer interface. The dethia analogues fully activate SaPC but demonstrate significantly reduced binding affinities relative to acetyl-CoA. Residues Arg₂₁, Lys₄₆, and Glu₄₁₈ of SaPC are located at the biotin carboxylase dimer interface and play a critical role in both allosteric activation and inhibition. A structure of R21A SaPC in complex with acetyl-CoA reveals an intact molecule of acetyl-CoA bound at the allosteric site, offering new molecular insights into the acetyl-CoA binding site. This study demonstrates that the biotin carboxylase domain dimer interface is a critical allosteric site in PC, serving as a convergence point for allosteric activation by acetyl-CoA and inhibition by l-aspartate.

Comments

Accepted version. Biochemistry, Vol. 62, No. 17 (2023): 2632-2644. DOI. © 2023 American Chemical Society. Used with permission.

Recommended Citation

Laseke, Amanda J.; Boram, Trevor J.; Schneider, Nicholas O.; Lohman, Jeremy R.; and St. Maurice, Martin, "Allosteric Site at the Biotin Carboxylase Dimer Interface Mediates Activation and Inhibition in Staphylococcus aureus Pyruvate Carboxylase" (2023). Biological Sciences Faculty Research and Publications. 964.
https://epublications.marquette.edu/bio_fac/964