SUMMARY Cyclic di-adenosine monophosphate (c-di-AMP) is a broadly conserved second messenger required for bacterial growth and infection. However, the molecular mechanisms of c-di-AMP signaling are still poorly understood. Using a chemical proteomics screen for c-di-AMP interacting proteins in the pathogen Listeria monocytogenes, we identified several broadly conserved protein receptors, including the central metabolic enzyme pyruvate carboxylase (LmPC). Biochemical and crystallographic studies of the LmPC-c-di-AMP interaction revealed a previously unrecognized allosteric regulatory site 25 Å from the active site. Mutations in this site disrupted c-di-AMP binding and affected enzyme catalysis of LmPC as well as PC from pathogenic Enterococcus faecalis. C-di-AMP depletion resulted in altered metabolic activity in L. monocytogenes. Correction of this metabolic imbalance rescued bacterial growth, reduced bacterial lysis, and resulted in enhanced bacterial burdens during infection. These findings greatly expand the c-di-AMP signaling repertoire and reveal a central metabolic regulatory role for a cyclic di-nucleotide.

Messenger RNA precursors (pre-mRNAs) are produced as the nascent transcripts of RNA polymerase II (Pol II) in eukaryotes and must undergo extensive maturational processing, including 5′-end capping, splicing, and 3′-end cleavage and polyadenylation. This review will summarize the structural and functional information reported over the past few years on the large machinery required for the 3′-end processing of most pre-mRNAs, as well as the distinct machinery for the 3′-end processing of replication-dependent histone pre-mRNAs, which have provided great insights into the proteins and their subcomplexes in these machineries. Structural and biochemical studies have also led to the identification of a new class of enzymes (the DXO family enzymes) with activity toward intermediates of the 5′-end capping pathway. Functional studies demonstrate that these enzymes are part of a novel quality surveillance mechanism for pre-mRNA 5′-end capping. Incompletely capped pre-mRNAs are produced in yeast and human cells, in contrast to the general belief in the field that capping always proceeds to completion, and incomplete capping leads to defects in splicing and 3′-end cleavage in human cells. The DXO family enzymes are required for the detection and degradation of these defective RNAs.

Geranyl-CoA carboxylase (GCC) is essential for the growth of Pseudomonas organisms with geranic acid as the sole carbon source. GCC has the same domain organization and shares strong sequence conservation with the related biotin-dependent carboxylases 3-methylcrotonyl-CoA carboxylase (MCC) and propionyl-CoA carboxylase (PCC). Here we report the crystal structure of the 750-kDa α6β6 holoenzyme of GCC, which is similar to MCC but strikingly different from PCC. The structures provide evidence in support of two distinct lineages of biotin-dependent acyl-CoA carboxylases, one carboxylating the α carbon of a saturated organic acid and the other carboxylating the γ carbon of an α-β unsaturated acid. Structural differences in the active site region of GCC and MCC explain their distinct substrate preferences. Especially, a glycine residue in GCC is replaced by phenylalanine in MCC, which blocks access by the larger geranyl-CoA substrate. Mutation of this residue in the two enzymes can change their substrate preferences.

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