An archaeal ADP-dependent serine kinase involved in cysteine biosynthesis and serine metabolism

Abstract

Routes for cysteine biosynthesis are still unknown in many archaea. Here we find that the hyperthermophilic archaeon Thermococcus kodakarensis generates cysteine from serine via O-phosphoserine, in addition to the classical route from 3-phosphoglycerate. The protein responsible for serine phosphorylation is encoded by TK0378, annotated as a chromosome partitioning protein ParB. The TK0378 protein utilizes ADP as the phosphate donor, but in contrast to previously reported ADP-dependent kinases, recognizes a non-sugar substrate. Activity is specific towards free serine, and not observed with threonine, homoserine and serine residues within a peptide. Genetic analyses suggest that TK0378 is involved in serine assimilation and clearly responsible for cysteine biosynthesis from serine. TK0378 homologs, present in Thermococcales and Desulfurococcales, are most likely not ParB proteins and constitute a group of kinases involved in serine utilization.

Figure 1. The three previously known pathways for Cys and Ser biosyntheses.

(a) The three pathways for Cys biosynthesis (Pathway Cys1, Cys2 and Cys3) are highlighted with grey arrows. In some bacteria, O-phosphohomoserine is replaced by O-acetylhomoserine. (b) The three previously known pathways for Ser biosynthesis (Pathway Ser1, Ser2 and Ser3) are highlighted with grey arrows. Compound abbreviations: 2-OG, 2-oxoglutarate, Pi, phosphate; THF, tetrahydrofolate. Enzyme abbreviations: CBL, cystathionine β-lyase; CBS, cystathionine β-synthase; CGL, cystathionine γ-lyase; CGS, cystathionine γ-synthase; CysK, cysteine synthase; GlyA, glycine/serine hydroxymethyltransferase; Pat, phosphoserine aminotransferase; PHSS, phosphohomoserine sulfhydrylase; SAT, serine acetyltransferase.

Figure 2. Cys or Met auxotrophy of Thermococcus kodakarensis gene disruptants.

(a,b,e–h) Cys auxotrophy was investigated by cultivating cells in ASW-AA-S⁰-Ura⁺ medium with (a,e,g) or without Cys (b,f,h). Symbols: T. kodakarensis KU216 (open circles), Δtk1449-2-1-2 (open squares), ΔserAΔldhA1ΔldhA2-1 (open triangles), Δtk1449ΔserAΔldhA1ΔldhA2-2 (open diamonds), ΔcysK-3 (black circles), ΔserK-1 (grey circles), Δtk1449ΔserK-1 (grey squares) and ΔserAΔldhA1ΔldhA2ΔserK-1 (black triangles). (c,d) Met auxotrophy was examined by cultivating cells in ASW-AA-S⁰-Ura⁺ medium with (c) or without Met (d). Symbols: T. kodakarensis KU216 (open circles) and Δtk1449-2-1-2 (open squares). Error bars indicate the standard deviations of three independent culture experiments.

Figure 3. ADP-dependent Ser kinase activity and substrate specificity of the TK0378 protein.

ADP-dependent Ser kinase activity of recombinant TK0378 protein was examined. Phosphate acceptor specificity (a,c) and phosphate donor specificity (b,d) were investigated. (a) Grey line, no phosphate acceptor; black solid line, 50 mM Ser; black broken line, 50 mM Thr; black dotted line, 50 mM homoserine. (b) Black solid line, 20 mM ADP; black broken line, 20 mM ATP; grey line, 1 mM standard Sep. (c) Grey line, no substrate; black solid line, 50 mM Ser; black broken line, 100 μM Crossitide (a substrate for Ser protein kinase). (d) As 20 mM pyrophosphate precipitated in reaction mixture, phosphate donors were applied here at 0.5 mM. Black solid line, 0.5 mM ADP; black broken line, 0.5 mM pyrophosphate; grey line, 1 mM standard O-phosphoserine (Sep).

Figure 4. Predicted pathways for Cys and Ser metabolism in Thermococcus kodakarensis.

Pathways involved in Cys biosynthesis, Ser biosynthesis and Ser assimilation identified in this study are highlighted with blue, grey and pink arrows, respectively. Cys can be generated from Ser or 3-phosphoglycerate. Ser can be generated from Thr (via Gly) or 3-phosphoglycerate. Ser can be assimilated to 3-phosphoglycerate. The SerK reaction is indicated with a thick arrow. THF, tetrahydrofolate.

Figure 5. Ser, Gly or Cys auxotrophy of Thermococcus kodakarensis gene disruptants.

Ser or Gly auxotrophy was investigated by cultivating cells in synthetic amino acid medium with (a) or without Ser (b) or with (c) or without Gly (d). Symbols: T. kodakarensis KU216 (open circles), ΔserAΔldhA1ΔldhA2-1 (open triangles), ΔglyA (open squares), Δtdh (grey triangles), ΔglyAΔtdh (black squares). Contribution of the route from 3-phosphoglycerate to Ser biosynthesis was examined in synthetic amino acid medium supplemented with pyruvate and depleted of Ser (e). Contribution of the route from 3-phosphoglycerate to Cys biosynthesis was examined in synthetic amino acid medium depleted of Cys with or without pyruvate (f). (e) Symbols: ΔglyA (open squares), ΔglyAΔserA (black circles), ΔglyAΔldhA1 (grey diamonds), ΔglyAΔldhA2 (black diamonds). (f) Symbols: KU216 host strain (open circles), ΔserK (grey circles), KU216 host strain with pyruvate (grey squares), ΔserK with pyruvate (black triangles). Error bars indicate the standard deviations of three independent culture experiments.