The Replicative DnaE Polymerase of Bacillus subtilis Recruits the Glycolytic Pyruvate Kinase (PykA) When Bound to Primed DNA Templates

Abstract

The glycolytic enzyme PykA has been reported to drive the metabolic control of replication through a mechanism involving PykA moonlighting functions on the essential DnaE polymerase, the DnaC helicase and regulatory determinants of PykA catalytic activity in Bacillus subtilis. The mutants of this control suffer from critical replication and cell cycle defects, showing that the metabolic control of replication plays important functions in the overall rate of replication. Using biochemical approaches, we demonstrate here that PykA interacts with DnaE for modulating its activity when the replication enzyme is bound to a primed DNA template. This interaction is mediated by the CAT domain of PykA and possibly allosterically regulated by its PEPut domain, which also operates as a potent regulator of PykA catalytic activity. Furthermore, using fluorescence microscopy we show that the CAT and PEPut domains are important for the spatial localization of origins and replication forks, independently of their function in PykA catalytic activity. Collectively, our data suggest that the metabolic control of replication depends on the recruitment of PykA by DnaE at sites of DNA synthesis. This recruitment is likely highly dynamic, as DnaE is frequently recruited to and released from replication machineries to extend the several thousand RNA primers generated from replication initiation to termination. This implies that PykA and DnaE continuously associate and dissociate at replication machineries for ensuring a highly dynamic coordination of the replication rate with metabolism.

Open Questions about the Roles of DnaA, Related Proteins, and Hyperstructure Dynamics in the Cell Cycle

“…Another way is via a central carbon metabolism that, in E. coli , can: (1) promote DnaA to its active DnaA-ATP form and its binding to oriC by cAMP (a regulator of this part of metabolism) [ 49 ]; (2) suppress the defects of the dnaA46 mutant by changes in pyruvate and acetate metabolism [ 50 ]; (3) inhibit DnaA conversion to DnaA-ATP and its binding to oriC (via acetylation of DnaA, with acetyl-CoA and acetyl-phosphate as donors) (for references see [ 51 ]). Evidence for the involvement of the central carbon metabolism in DNA replication in Bacillus subtilis includes: (1) subunits of pyruvate dehydrogenase (PdhC) and related enzymes bind the origin of replication region, DnaC and DnaG inhibit the initiation of replication; (2) mutations in the genes of central carbon metabolism suppress initiation and elongation defects in dnaC , dnaG , and dnaE mutants; (3) mutations in gapA (which encodes glyceraldehyde 3-phosphate dehydrogenase) perturb the metabolic control of replication; (4) pyruvate kinase (PykA) can both inhibit initiation and stimulate elongation via proposed interactions with DnaC, DnaG, and DnaE as modulated, perhaps, by phosphorylation [ 51 , 52 ].…”

The metabolic control of DNA replication: mechanism and function

“…The PEPut domain of PykA is not required for royalsocietypublishing.org/journal/rsob Open Biol. 13: 230220 the catalytic activity of the enzyme [67,107]. However, it interacts with CAT via a hydrogen bond between E208 (CAT) and L536 (PEPut), as shown in a structural study of the highly homologous (72.5% identity) Geobacillus stearothermophilus PykA [108].…”

“…when the CAT-PEPut interaction is impeded) (figure 4, right panel). In addition to these distinct effects of PykA conformers on initiation, it was shown that (i) PykA physically interacts with the polymerase DnaE via its CAT domain when the replication enzyme is bound to primed DNA [67], (ii) PEPut modulates the strength of this interaction and its effect on the DnaE polymerase activity [66,67], and (iii) DnaE is early recruited at replication origins during initiation [61]. We thus suggest from these data that However, while the T-state conformer dramatically increases DnaE activity and causes early initiation, the R-state conformer allows a proper polymerase activity and initiation timing.…”

“…In these enzymes, the PEP utilizer moves around a flexible arm [ 98 , 99 ] and is phosphorylated at a conserved TSH motif at the expense of phosphoenolpyruvate and ATP to drive sugar imports and catalytic or regulatory activities [ 98 , 100 – 106 ]. The PEPut domain of PykA is not required for the catalytic activity of the enzyme [ 67 , 107 ]. However, it interacts with CAT via a hydrogen bond between E208 (CAT) and L536 (PEPut), as shown in a structural study of the highly homologous (72.5% identity) Geobacillus stearothermophilus PykA [ 108 ].…”

“…However, it interacts with CAT via a hydrogen bond between E208 (CAT) and L536 (PEPut), as shown in a structural study of the highly homologous (72.5% identity) Geobacillus stearothermophilus PykA [ 108 ]. In B. subtilis , this interaction seems to stimulate PykA activity and to be negatively regulated by phosphorylation of the T residue of the TSH motif which maps immediately downstream of the interacting L536 amino acid (residues 537–539) [ 66 , 67 ]. An AlphaFold2 analysis predicts two B. subtilis PykA monomer structures with a highly similar spatial CAT domain and alternative PEPut positions ( figure 3 , top left panel) [ 109 , 110 ], indicating that PEPut is carried by a flexible arm, as in other PEP utilizer containing metabolic enzymes [ 98 , 99 ].…”