Removal of phosphoglycolate in hyperthermophilic archaea

Abstract

Many organisms that utilize the Calvin-Benson-Bassham (CBB) cycle for autotrophic growth harbor metabolic pathways to remove and/or salvage 2-phosphoglycolate, the product of the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). It has been presumed that the occurrence of 2-phosphoglycolate salvage is linked to the CBB cycle, and in particular, the C2 pathway to the CBB cycle and oxygenic photosynthesis. Here, we examined 2-phosphoglycolate salvage in the hyperthermophilic archaeon Thermococcus kodakarensis, an obligate anaerobe that harbors a Rubisco that functions in the pentose bisphosphate pathway. T. kodakarensis harbors enzymes that have the potential to convert 2-phosphoglycolate to glycine and serine, and their genes were identified by biochemical and/or genetic analyses. 2-phosphoglycolate phosphatase activity increased 1.6-fold when cells were grown under microaerobic conditions compared to anaerobic conditions. Among two candidates, TK1734 encoded a phosphatase specific for 2-phosphoglycolate, and the enzyme was responsible for 80% of the 2-phosphoglycolate phosphatase activity in T. kodakarensis cells. The TK1734 disruption strain displayed growth impairment under microaerobic conditions, which was relieved upon addition of sodium sulfide. In addition, glycolate was detected in the medium when T. kodakarensis was grown under microaerobic conditions. The results suggest that T. kodakarensis removes 2-phosphoglycolate via a phosphatase reaction followed by secretion of glycolate to the medium. As the Rubisco in T. kodakarensis functions in the pentose bisphosphate pathway and not in the CBB cycle, mechanisms to remove 2-phosphoglycolate in this archaeon emerged independent of the CBB cycle.

Keywords: 2-phosphoglycolate; Archaea; Rubisco; metabolism.

Fig. 1.

The classical C2 pathway and 2-PG removal in Thermococcus kodakarensis. (A) The classical photorespiration pathways in plants, algae, and cyanobacteria consist of 2-PG phosphatase (PGP), GOX or glycolate dehydrogenase (GLDH), alanine:glyoxylate aminotransferase (AGAT), glutamate:glyoxylate aminotransferase (GGAT) and/or serine:glyoxylate aminotransferase (SGAT), serine hydroxymethyltransferase (GlyA), glycine cleavage system (GCS), serine:2-oxoglutarate aminotransferase or SGAT, hydroxypyruvate reductase (HPR), and GLYK. (B) 2-PG removal/salvage proposed in T. kodakarensis. Dephosphorylation of 2-PG and excretion of glycolate have been experimentally verified. GLDH that catalyzes the oxidation of glycolate is not a homolog of previously identified GLDH enzymes, and is a member of the lactate dehydrogenase family. Activity levels of GLDH in T. kodakarensis cells are much lower than those of other enzymes, and the conversion is thus indicated by a dotted arrow. The enzymes in a potential route from serine to 3-phosphoglycerate are serine kinase (SerK), phosphoserine aminotransferase (PAT), and 3-phosphoglycerate dehydrogenase (SerA). Distinct to the classical C2 pathway, phosphorylation precedes transamination and reduction. Genes and proteins experimentally validated in this study are indicated in bold.

Fig. 2.

Detection of enzyme activity in T. kodakarensis cell extracts. (A) 2-PG phosphatase activity in cell extracts from T. kodakarensis KU216, ΔTK1734, and ΔTK2301 strains. Gray or white bars indicate the specific activities in extracts from cells grown under anaerobic or microaerobic conditions, respectively. (B) GLDH activity from glycolate to glyoxylate in cell extracts from KU216, ΔTK0551, and ΔTK0683 strains. Gray or white bars indicate the specific activities in extracts from cells grown under anaerobic or microaerobic conditions, respectively. (C) GLDH activity from glyoxylate to glycolate in cell extracts from KU216, ΔTK0551, and ΔTK0683 strains. Gray or white bars indicate the specific activities in extracts from cells grown under anaerobic or microaerobic conditions, respectively. (D) AGAT activity in cell extracts from KU216, ΔTK0186, ΔTK0548, ΔTK2268, and ΔTK1094 strains. Gray or white bars indicate the specific activities in extracts from cells grown under anaerobic or microaerobic conditions, respectively. Activity measurements were carried out as described in the Methods section at 80 °C. The data represent the average of three independent experiments and are shown with the SD values. 0.01 < P ≤ 0.05*; 10⁻³ < P ≤ 10⁻²**; 10⁻⁴ < P ≤ 10⁻³***; 10⁻⁵ < P ≤ 10⁻⁴****; 10⁻⁷ < P ≤ 10⁻⁶******; 10⁻⁸ < P ≤ 10⁻⁷*******.

Fig. 3.

Phosphatase, dehydrogenase, or aminotransferase activity of recombinant proteins with various substrates. Phosphatase activities of the purified TK1734 (A) and TK2301 (B) proteins were measured toward various phosphorylated metabolites. Dehydrogenase activities of the purified TK0551 (C) and TK0683 (D) proteins were examined toward various 2-hydroxyacids. Aminotransferase activities of the purified TK0186/TK1094 proteins with various amino donor compounds in the presence of glyoxylate as the amino acceptor (E/G) or various amino acceptor compounds in the presence of alanine as the amino donor (F/H) were evaluated. In all cases (A–H), activity measurements were carried out as described in the Methods section at 80 °C. ND: not detected. The data represent the average of three independent experiments and are shown with the SD values.

Fig. 4.

Growth properties of T. kodakarensis KU216 and ΔTK0150 strains. Serine auxotrophy was examined by cultivating cells in ASW-AA-S⁰-Ura⁺ medium with or without serine. Symbols: KU216 with (closed circles) or without serine (closed squares), ΔTK0150 with (open circles) or without (open squares) serine. Error bars indicate the SD values of three independent culture experiments.

Fig. 5.

Growth properties of T. kodakarensis KU216 and ΔTK1734 strains under microaerobic conditions. KU216 (closed circles) and ΔTK1734 (open circles) strains were cultivated in ASW-YT-m1-Pyr medium. All media were left in an anaerobic box for 24 h prior to inoculation to allow dissolved gases to equilibrate with the atmosphere in the anaerobic box (oxygen concentration: ~0.1%). Media for (A) were prepared without Na₂S (microaerobic conditions), and the concentration of Na₂S in (B–D) were 0.08 mM, 0.16 mM, and 0.32 mM, respectively. Error bars indicate the SD values of three independent culture experiments.