Glycerate 2-kinase of Thermotoga maritima and genomic reconstruction of related metabolic pathways.
Members of a novel glycerate-2-kinase (GK-II) family were tentatively identified in a broad range of species, including eukaryotes and archaea and many bacteria that lack a canonical enzyme of the GarK (GK-I) family. The recently reported three-dimensional structure of GK-II from Thermotoga maritima (TM1585; PDB code 2b8n) revealed a new fold distinct from other known kinase families. Here, we verified the enzymatic activity of TM1585, assessed its kinetic characteristics, and used directed mutagenesis to confirm the essential role of the two active-site residues Lys-47 and Arg-325. The main objective of this study was to apply comparative genomics for the reconstruction of metabolic pathways associated with GK-II in all bacteria and, in particular, in T. maritima. Comparative analyses of approximately 400 bacterial genomes revealed a remarkable variety of pathways that lead to GK-II-driven utilization of glycerate via a glycolysis/gluconeogenesis route. In the case of T. maritima, a three-step serine degradation pathway was inferred based on the tentative identification of two additional enzymes, serine-pyruvate aminotransferase and hydroxypyruvate reductase (TM1400 and TM1401, respectively), that convert serine to glycerate via hydroxypyruvate. Both enzymatic activities were experimentally verified, and the entire pathway was validated by its in vitro reconstitution.
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D019295:Computational Biology; D016680:Genome, Bacterial; D023281:Genomics / Q000379:methods; D005988:Glyceric Acids / Q000378:metabolism; D050537:Hydroxypyruvate Reductase / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D011773:Pyruvates / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D018085:Regulon / Q000235:genetics; D012694:Serine / Q000378:metabolism; D020124:Thermotoga maritima / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D000637:Transaminases / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D005988:Glyceric Acids; D011773:Pyruvates; D011993:Recombinant Fusion Proteins; D012694:Serine; C042971:glyceric acid; C012375:hydroxypyruvic acid; D050537:Hydroxypyruvate Reductase; D000637:Transaminases; C022638:serine-pyruvate aminotransferase; D017853:Phosphotransferases (Alcohol Group Acceptor); C020670:glycerate kinase