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

Proceedings of the National Academy of Sciences of the United States of America (2017-08-16)
Philip H Choi, Thu Minh Ngoc Vu, Huong Thi Pham, Joshua J Woodward, Mark S Turner, Liang Tong
ABSTRACT

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. Our earlier studies showed that c-di-AMP regulates central metabolism in Listeria monocytogenes by inhibiting its pyruvate carboxylase (LmPC), a biotin-dependent enzyme with biotin carboxylase (BC) and carboxyltransferase (CT) activities. We report here structural, biochemical, and functional studies on the inhibition of Lactococcus lactis PC (LlPC) by c-di-AMP. The compound is bound at the dimer interface of the CT domain, at a site equivalent to that in LmPC, although it has a distinct binding mode in the LlPC complex. This binding site is not well conserved among PCs, and only a subset of these bacterial enzymes are sensitive to c-di-AMP. Conformational changes in the CT dimer induced by c-di-AMP binding may be the molecular mechanism for its inhibitory activity. Mutations of residues in the binding site can abolish c-di-AMP inhibition. In L. lactis, 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 expression of a c-di-AMP-insensitive LlPC. LlPC has high intrinsic catalytic activity and is not sensitive to acetyl-CoA activation, in contrast to other PC enzymes.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Dulbecco′s Modified Eagle′s Medium - low glucose, With 1000 mg/L glucose, and sodium bicarbonate, without L-glutamine and phenol red, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Aspartate Assay Kit, sufficient for 100 colorimetric or fluorometric tests