Structural and functional studies of pyruvate carboxylase regulation by cyclic di-AMP in lactic acid bacteria

Significance Cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) is a broadly conserved bacterial second messenger that has been implicated in a wide range of cellular processes. We report here structural, biochemical, and functional studies on the inhibition of Lactococcus lactis pyruvate carboxylase (LlPC) by c-di-AMP. The compound has a distinct binding mode in LlPC compared with that in Listeria monocytogenes PC. Mutations of residues in the binding site can abolish c-di-AMP inhibition. LlPC is required for efficient milk acidification through its essential role in aspartate biosynthesis. The aspartate pool in L. lactis is negatively regulated by c-di-AMP, and high aspartate levels can be restored by a c-di-AMP–insensitive LlPC. LlPC has high intrinsic catalytic activity and is insensitive to acetyl-CoA activation, in contrast to other PCs.