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

Allosteric regulation of the essential anaplerotic enzyme,pyruvatecarboxylase (PC), is vital for metabolic homeostasis. PC catalyzesthe bicarbonate- and ATP-dependent carboxylation of pyruvate to formoxaloacetate. Dysregulation of PC activity can impact glucose andredox metabolism, which contributes to the pathogenicity of many diseases.To maintain homeostasis, PC is allosterically activated by acetyl-CoAand allosterically inhibited by l-aspartate. In this study,we further characterize the molecular basis of allosteric regulationin Staphylococcus aureus PC (SaPC) using slowly/nonhydrolyzable dethia analogues of acetyl-CoAand site-directed mutagenesis of residues at the biotin carboxylasehomodimer interface. The dethia analogues fully activate SaPC but demonstrate significantly reduced binding affinities relativeto acetyl-CoA. Residues Arg(21), Lys(46), and Glu(418) of SaPC are located at the biotin carboxylasedimer interface and play a critical role in both allosteric activationand inhibition. A structure of R21A SaPC in complexwith acetyl-CoA reveals an intact molecule of acetyl-CoA bound atthe allosteric site, offering new molecular insights into the acetyl-CoAbinding site. This study demonstrates that the biotin carboxylasedomain dimer interface is a critical allosteric site in PC, servingas a convergence point for allosteric activation by acetyl-CoA andinhibition by l-aspartate.

Automated summary

This study further characterizes the allosteric regulation of Staphylococcus aureus pyruvatecarboxylase (SaPC), revealing the critical role of Arg(21), Lys(46), and Glu(418) residues in the biotin carboxylasedimer interface in the allosteric activation and inhibition of SaPC. The structure of R21A SaPC in complex with acetyl-CoA provides new insights into the acetyl-CoA binding site and its regulatory function.