Identification and Enzymatic Analysis of an Archaeal ATP-Dependent Serine Kinase from the Hyperthermophilic Archaeon Staphylothermus marinus

Abstract

Serine kinase catalyzes the phosphorylation of free serine (Ser) to produce O-phosphoserine (Sep). An ADP-dependent Ser kinase in the hyperthermophilic archaeon Thermococcus kodakarensis (Tk-SerK) is involved in cysteine (Cys) biosynthesis and most likely Ser assimilation. An ATP-dependent Ser kinase in the mesophilic bacterium Staphylococcus aureus is involved in siderophore biosynthesis. Although proteins displaying various degrees of similarity with Tk-SerK are distributed in a wide range of organisms, it is unclear if they are actually Ser kinases. Here, we examined proteins from Desulfurococcales species in Crenarchaeota that display moderate similarity with Tk-SerK from Euryarchaeota (42 to 45% identical). Tk-serK homologs from Staphylothermus marinus (Smar_0555), Desulfurococcus amylolyticus (DKAM_0858), and Desulfurococcus mucosus (Desmu_0904) were expressed in Escherichia coli. All three partially purified recombinant proteins exhibited Ser kinase activity utilizing ATP rather than ADP as a phosphate donor. Purified Smar_0555 protein displayed activity for l-Ser but not other compounds, including d-Ser, l-threonine, and l-homoserine. The enzyme utilized ATP, UTP, GTP, CTP, and the inorganic polyphosphates triphosphate and tetraphosphate as phosphate donors. Kinetic analysis indicated that the Smar_0555 protein preferred nucleoside 5'-triphosphates over triphosphate as a phosphate donor. Transcript levels and Ser kinase activity in S. marinus cells grown with or without serine suggested that the Smar_0555 gene is constitutively expressed. The genes encoding Ser kinases examined here form an operon with genes most likely responsible for the conversion between Sep and 3-phosphoglycerate of central sugar metabolism, suggesting that the ATP-dependent Ser kinases from Desulfurococcales play a role in the assimilation of Ser. IMPORTANCE Homologs of the ADP-dependent Ser kinase from the archaeon Thermococcus kodakarensis (Tk-SerK) include representatives from all three domains of life. The results of this study show that even homologs from the archaeal order Desulfurococcales, which are the most structurally related to the ADP-dependent Ser kinases from the Thermococcales, are Ser kinases that utilize ATP, and in at least some cases inorganic polyphosphates, as the phosphate donor. The differences in properties between the Desulfurococcales and Thermococcales enzymes raise the possibility that Tk-SerK homologs constitute a group of kinases that phosphorylate free serine with a wide range of phosphate donors.

Keywords: Archaea; Crenarchaea; Staphylothermus; enzymes; hyperthermophile; metabolism; serine kinase.

FIG 1

Phylogenetic analysis of Tk-SerK and Sa-SbnI homologs. Homologs of Tk-SerK (tko:TK0378) and Sa-SbnI (saa:SAUSA300_0126) were collected and examined as described in the text. The phylogenetic tree was constructed using fasttree. SH-like local support values are indicated at the nodes. Black letters with circles, blue letters with squares, and red letters with stars indicate proteins from Archaea, Bacteria, and Eukarya, respectively. The colors of the symbols indicate the taxonomic orders shown in the upper right corner. Red, green, and blue bars indicate the subgroups including an archaeal ADP-dependent Ser kinase (9, 20), a bacterial ATP-dependent Ser kinase (13, 14), and an archaeal ATP-dependent Ser kinase identified in this study, respectively. Each protein is indicated with a three- or four-letter organism code used in the KEGG BLAST search, followed by the locus tag. Organism codes used in this figure are defined in Fig. S1. Thermococci and Thermoprotei are classified as Euryarchaeota and Crenarchaeota, respectively.

FIG 2

Examination of Ser kinase activity of Tk-SerK homologs in Desulfurococcales. (A) ADP-dependent Ser kinase activity was examined by detecting AMP production. ATP-dependent Ser kinase activity was investigated by detecting ADP production (B) and Sep production (C). (D) Negative control with the cell extract from E. coli transformed with an empty vector pET21a(+). In all panels, black, red, green, blue, and yellow lines indicate reaction mixtures including no protein, Smar_0555, DKAM_0858, Desmu_0904, and the cell extract from E. coli with an empty vector, respectively. Dashed lines show each standard compound, AMP (A), ADP (B), and Sep (C and D). a.u., arbitrary units.

FIG 3

Substrate specificity of the Smar_0555 protein. (A) Phosphate donor specificity was examined in the presence of 60 mM Ser and 40 mM each phosphate donor substrate at 85°C. Specific activity was determined by quantifying the produced NDPs with coupling enzymes and the produced Sep with HPLC when NTPs and polyphosphates were used as phosphate donors, respectively. (B) Phosphate acceptor specificity was investigated in the presence of 40 mM ATP and a 60 mM concentration of each substrate at 85°C. Specific activity was measured by quantifying the ADP produced. The activities are calculated from three independent experiments. Error bars indicate standard deviations. ND, not detected.

FIG 4

Divalent metal ion requirement of ATP-dependent Ser kinase activity of Sm-SerK. Each metal ion was added at a concentration of 20 mM, and the reactions were carried out with 50 mM Ser and 20 mM ATP at 85°C and pH 7.5. The activities were measured by quantifying the ADP produced and were calculated from three independent experiments. Error bars indicate standard deviations. ND, not detected.

FIG 5

Temperature and pH dependencies of Sm-SerK. (A) ATP-dependent kinase activity was measured with 50 mM Ser and 20 mM ATP at various temperatures between 15 and 95°C at pH 8.0. (B) Arrhenius plot of the data shown in panel A. (C) The pH dependency of the ATP-dependent kinase at 85°C was examined with 50 mM Ser and 20 mM ATP at various pH values between 6.5 and 11 using the following buffers: PIPES-NaOH (pH 6.5 to 7.5) (filled diamonds), Tris-HCl (pH 7.5 to 8.0) (open squares), bicine-NaOH (pH 8.0 to 9.0) (triangles), CHES-NaOH (pH 9.0 to 10.0) (open diamonds), and CAPS-NaOH (pH 10.0 to 11.0) (filled squares).

FIG 6

Kinetic analysis of the Sm-SerK reaction. Initial velocities at 15°C with various concentrations of Ser (A), ATP (B), and UTP (C) were determined, while initial velocities at various concentrations of PPPi were examined at 85°C (D).

FIG 7

Sequence alignment of Ser kinases from Desulfurococcales with Tk-SerK and Sa-SbnI. Protein sequences from Desulfurococcales are Smar_0555 (Sm-SerK), DKAM_0858, and Desmu_0904. Arrowheads in the Tk-SerK row indicate residues identified through structural analysis of the enzyme (20). Arrowheads in the Sa-SbnI row indicate residues predicted based on mutational analysis of Sa-SbnI and crystallographic analysis of Tk-SerK, Sa-SbnI, and the SbnI protein from Staphylococcus pseudintermedius (13, 14, 20). The colors represent the heme iron-coordinating ligands (gray), the catalytic residue (red), residues recognizing the adenine and ribose moieties of AMP/ADP (blue), the magnesium ion (black), and the phosphate group of AMP/ADP/Sep in Tk-SerK or ADP/ATP/Sep in Sa-SbnI (green), and residues interacting with the Ser moiety of Sep (orange). Residues highlighted in cyan indicate conserved residues among the Ser kinases from Desulfurococcales.