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

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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.

No MeSH data available.


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.
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f2: 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.

Mentions: In order to identify the pathway(s) involved in Cys biosynthesis in T. kodakarensis, we disrupted genes presumed to encode the enzymes of each previously identified pathway. After isolating the transformants, genotypes were confirmed by PCR (Supplementary Figs 2 and 3, Supplementary Table 1). We first examined the involvement of pathway Cys1 (Fig. 1a), which consists of cystathionine β-synthase (CBS) and CGL. There was not a very strong candidate for a CBS in T. kodakarensis. The TK1687 protein is 34% identical to the N-terminal region of the CBS from Saccharomyces cerevisiae S288c (507 residues), but is much smaller in size (273 residues). On the other hand, the TK1687 protein is similar in both size and primary structure to the archaeal cysteine synthases (CysK) from Methanosarcina thermophila (37% identical) and A. pernix (36%). Concerning CGL, the TK1449 protein product is 38% identical to the CGL from S. cerevisiae S288c. Other genes with notable similarity to CGL were not found on the genome. In order to examine the possibility that TK1449 encodes a CGL and that Cys is synthesized via pathway Cys1, we constructed a TK1449 gene disruption strain. Growth of the Δtk1449 mutant in a synthetic medium without Cys was similar to that of the host strain (Fig. 2a,b), suggesting that the main pathway for Cys biosynthesis is not pathway Cys1. On the other hand, the TK1449 protein is also 25% identical to cystathionine β-lyase (CBL) and 35% identical to cystathionine γ-synthase (CGS) from E. coli, which function for Met biosynthesis. When we examined the effects of TK1449 disruption on Met biosynthesis in T. kodakarensis, we observed clear Met auxotrophy (Fig. 2c,d), suggesting that this gene is involved in Met biosynthesis, most likely encoding a CGS and/or a CBL, involved in the conversion of O-phosphohomoserine to L-homocysteine via L-cystathionine (Fig. 1a). The TK1449 protein may also function as a phosphohomoserine sulfhydrylase, an activity detected in plants that directly converts O-phosphohomoserine to L-homocysteine (Fig. 1a).


An archaeal ADP-dependent serine kinase involved in cysteine biosynthesis and serine metabolism
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.
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f2: 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.
Mentions: In order to identify the pathway(s) involved in Cys biosynthesis in T. kodakarensis, we disrupted genes presumed to encode the enzymes of each previously identified pathway. After isolating the transformants, genotypes were confirmed by PCR (Supplementary Figs 2 and 3, Supplementary Table 1). We first examined the involvement of pathway Cys1 (Fig. 1a), which consists of cystathionine β-synthase (CBS) and CGL. There was not a very strong candidate for a CBS in T. kodakarensis. The TK1687 protein is 34% identical to the N-terminal region of the CBS from Saccharomyces cerevisiae S288c (507 residues), but is much smaller in size (273 residues). On the other hand, the TK1687 protein is similar in both size and primary structure to the archaeal cysteine synthases (CysK) from Methanosarcina thermophila (37% identical) and A. pernix (36%). Concerning CGL, the TK1449 protein product is 38% identical to the CGL from S. cerevisiae S288c. Other genes with notable similarity to CGL were not found on the genome. In order to examine the possibility that TK1449 encodes a CGL and that Cys is synthesized via pathway Cys1, we constructed a TK1449 gene disruption strain. Growth of the Δtk1449 mutant in a synthetic medium without Cys was similar to that of the host strain (Fig. 2a,b), suggesting that the main pathway for Cys biosynthesis is not pathway Cys1. On the other hand, the TK1449 protein is also 25% identical to cystathionine β-lyase (CBL) and 35% identical to cystathionine γ-synthase (CGS) from E. coli, which function for Met biosynthesis. When we examined the effects of TK1449 disruption on Met biosynthesis in T. kodakarensis, we observed clear Met auxotrophy (Fig. 2c,d), suggesting that this gene is involved in Met biosynthesis, most likely encoding a CGS and/or a CBL, involved in the conversion of O-phosphohomoserine to L-homocysteine via L-cystathionine (Fig. 1a). The TK1449 protein may also function as a phosphohomoserine sulfhydrylase, an activity detected in plants that directly converts O-phosphohomoserine to L-homocysteine (Fig. 1a).

View Article: PubMed Central - PubMed

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.

No MeSH data available.