The mechanism underlying a unique response to acetyl CoA activation in eukaryotic pyruvate carboxylase from Aspergillus nidulans

Abstract

Pyruvate carboxylase (PC) is a multifunctional, allosterically regulated, biotin‐dependent enzyme that carboxylates pyruvate to oxaloacetate. The conversion of pyruvate to oxaloacetate is accomplished by coupling two half reactions that are catalyzed at spatially distinct active sites in the Biotin Carboxylase (BC) and Carboxyltransferase (CT) domains, through the movement of a carrier domain (Biotin Carboxyl Carrier Protein; BCCP). PC is a homotetramer, with two subunits contributing to each face of the tetramer, and with homodimerization mediated between the individual domains at each corner of the tetramer. Acetyl coenzyme A (acetyl CoA) is an allosteric activator of PC found in most species. However, PC from Aspergillus nidulans (AnPC) is unique in not being stimulated by acetyl CoA. Here, we provide a comprehensive structural and kinetic characterization of AnPC, and compare it with the PC from Rhizobium etli (RePC) that is highly sensitive to acetyl CoA stimulation, in order to better understand the mechanism of allosteric activation of PC by acetyl CoA.We determined the structure of AnPC by X‐ray crystallography at 3.0 Å resolution. In this structure, the BCCP domain was observed to occupy a unique position, suggesting that the carrier domain in AnPC may proceed through an alternative translocation pathway. Interestingly, despite the absence of activation by acetyl CoA in the overall reaction catalyzed by AnPC, the two half‐reactions were activated by acetyl CoA in AnPC. Kinetic studies with a series of acetyl CoA analogues suggests that AnPC binds with acetyl CoA at the conserved acetyl CoA binding site, while the acetyl group plays a less significant role in the activation of AnPC compared to RePC. Modification of the acyl group reduces the binding affinity of the analogues with AnPC but not the extent of activation.To further dissect the contributions made by the individual catalytic domains to the allosteric response in PC, we generated two chimeric enzymes (ReBC‐AnPC and AnBC‐RePC) by fusing the enzymes at the interface with the N‐terminal BC domain. The coupling efficiency and acetyl CoA sensitivity of these chimeras were determined, revealing that both of the chimeras were stimulated by acetyl CoA and that allosteric activation is largely determined by the BC domain.Most interestingly, negative cooperativity was observed for ATP cleavage in the BC domain of the acetyl CoA sensitive RePC. These observations are consistent with a ‘half‐the‐sites' reactivity mechanism and can be interpreted in light of recent cryo‐EM studies reported in Staphylococcus aureus PC1. In contrast, negative cooperativity was observed for ATP cleavage only in the presence of acetyl CoA for AnPC. The acetyl moiety of acetyl CoA is thought to bind at the BC‐domain dimer interface and is considered to be critically important for allosteric activation. Our structural and kinetic data with AnPC suggest that the BC‐domain dimer interface may differ between RePC and AnPC, contributing to the divergent responses to acetyl CoA activation..Support or Funding InformationThis work was supported by the National Institutes of Health grant GM117540.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.