Characterization of glycerate kinase (2-phosphoglycerate forming), a key enzyme of the nonphosphorylative Entner–Doudoroff pathway, from the thermoacidophilic euryarchaeonPicrophilus torridus

Abstract

Picrophilus torridus has been shown to degrade glucose via a nonphosphorylative Entner-Doudoroff (ED) pathway. Here we report the characterization of a key enzyme of this pathway, glycerate kinase (2-phosphoglycerate forming). The enzyme was purified 5,100-fold to homogeneity. The 95 kDa homodimeric protein catalyzed the ATP-dependent phosphorylation of glycerate specifically to 2-phosphoglycerate. The enzyme showed highest activity at 60 degrees C and pH 7.3, with ATP as phosphoryl donor and Mg(2+) as divalent cation. By MALDI-TOF analysis, ORF Pto1442 was identified in the genome of P. torridus as the encoding gene, designated gck. Homologs with high sequence identity were identified in the genomes of the archaea Thermoplasma and Sulfolobus spp. and Thermoproteus tenax, for which the operation of nonphosphorylative ED pathways, involving 2-phosphoglycerate forming glycerate kinases, has been proposed.

Carbohydrate Metabolism in Archaea: Current Insights into Unusual Enzymes and Pathways and Their Regulation

“…In Thermoplasma and Picrophilus, the resulting GA is further oxidized via GA dehydrogenase to glycerate. GA is then phosphorylated by glycerate kinase to yield 2PG and further converted to a second molecule of pyruvate by ENO and PK (248,(251)(252)(253)(254). Due to bypassing phosphorylation at the level of KDG and also subsequent GAP oxidation, and since GA oxidation to glycerate is not coupled to ATP formation, the net ATP yield of the npED branch is zero (1 ATP invested for glycerate phosphorylation, and 1 ATP gained in the PK-catalyzed reaction).…”

“…The class I GK enzymes (GK I superfamily [SCOP database]) are described mainly for Bacteria and for a few Eukarya, which are involved, e.g., in E. coli in allantoin (GK-1) as well as in glucarate and galactarate utilization (GK-2) (310). The crystal structure of the Neisseria meningitidis enzyme (PDB accession number 1TO6; PubMed identification is not available) has been solved and indicates no structural or sequence similarity to the class II enzymes (251,252,311). The class III GKs were first characterized for Arabidopsis thaliana, and homologs have been identified mainly in plants, some Cyanobacteria, and fungi, where they function in photorespiration or glycerol metabolism (312; for details, see reference 252).…”

Glycerate 2-Kinase of Thermotoga maritima and Genomic Reconstruction of Related Metabolic Pathways

“…ClustalX (30) and PHYLIP (7) were used to construct multiple protein alignments and phylogenetic trees by the maximum likelihood method (16). The GK box regulatory DNA motif was identified by an iterative signal detection procedure implemented in SignalX software (24); additional GK box regulatory sites were located in the Thermotoga genomes, using Genome Explorer software (17).…”

“…A deficiency of human GK-II activity leads to the hereditary disease Dglyceric aciduria (31), which was tentatively connected with alternative splicing of the GLYCTK gene (9). Recently, a GK-II family member from the archaeon Picrophilus torridus (encoded by the gck gene) was characterized as a key enzyme in the nonphosphorylating Entner-Doudoroff pathway, characteristic of thermoacidophilic archaea such as P. torridus and Thermoplasma acidophilum (24,25). The first 3D structure recently reported for a GK-II family protein, TM1585, from Thermotoga maritima (PDB code 2b8n) revealed a novel fold and allowed tentative mapping of the active-site area (2,29).…”

“…The first 3D structure recently reported for a GK-II family protein, TM1585, from Thermotoga maritima (PDB code 2b8n) revealed a novel fold and allowed tentative mapping of the active-site area (2,29). Two characterized members of the GK-II family from M. extorquens (3) and P. torridus (24) were assigned 2PG-forming activity. The third, structurally unrelated GK family (termed here GK-III and also known as GLYK) includes the recently characterized 3PG-forming enzyme from Arabidopsis thaliana (1) and its homologs in other plants, fungi, nearly all cyanobacteria, and a few other bacterial species.…”