Figure 5: Rationalizing functional roles of highly conserved residues in DgkA not directly involved in catalysis.

(a) The anionic carboxyl of Glu34_C is proposed to elevate the pKa of catalytic residue Glu69_C making it a stronger base for proton abstraction from the lipid (green protein carbons). Electron density (mesh) for alternative Glu34 conformers was observed in the XFEL data recorded at RT (purple carbons) that may enable a switch in pKa at Glu69. (b) Alternative conformations for Glu76_C in the XFEL structure. The first (Glu76_1) is in position to coordinate with Zn2. Black dashed lines correspond to distances 2.0–2.2 Å. The second (Glu76_2) has the coordinating oxygen at some distance (3.8 Å, green dashed line) from Zn2 where it may facilitate product release. The polyphosphate and zinc are superimposed from the ternary complex on the XFEL apo-structure. (c) Side-chain amide nitrogen of Asn72_C coordinates with carboxyls of Glu69_C and Glu76_C, both essential residues. By weakly interacting with γ-phosphate oxygens, the electrophilicity of the γ-phosphorus is elevated making it more reactive. Additionally, the amide may stabilize a transient bisubstrate by being positioned close to where the pentavalent intermediate is likely to form between the 1-OH of MAG1 and the γ-phosphate of ACP (dotted blue line). Likely interactions between MAG1 and the enzyme are indicated by dashed lines only. (d) Alternative conformations for Glu69 observed in the XFEL structure (magenta carbons) superimposed on the DgkA ternary structure (green carbons). The first conformation (Glu69_1) is like that seen in the ternary complex and interacts with MAG1. The second (Glu69_2) extends into the membrane and may guide lipid substrate to the active site. (e) An expanded view of Glu69 in d with electron density corresponding to alternative conformers seen in the XFEL structure superposed. (f) The methyl side chain of Ala30_C (red circle) is proposed to provide room for lipid substrate and product to pass between the start of SH_B and the top of H1_C that define the gateway into and out of the active site. The Cα–Cα distance from Ala30_C (red circle) to Ala13_B (blue circle) in asBC is 10.0 Å.