Molecular and functional characterization of D-3-phosphoglycerate dehydrogenase in the serine biosynthetic pathway of the hyperthermophilic archaeon Sulfolobus tokodaii.
A gene (ST1218) encoding a D-3-phosphoglycerate dehydrogenase (PGDH; EC 1.1.1.95) homolog was found in the genome of Sulfolobus tokodaii strain 7 by screening a database of enzymes likely to contribute to l-serine biosynthesis in hyperthermophilic archaea. After expressing the gene in Escherichia coli, the PGDH activity of the recombinant enzyme was assessed. Homogeneous PGDH was obtained using conventional chromatography steps, though during the purification an unexpected decline in enzyme activity was observed if the enzyme was stored in plastic tubes, but not in glass ones. The purified enzyme was a homodimer with a subunit molecular mass of about 35kDa and was highly thermostable. It preferably acted as an NAD-dependent D-3-phosphoglycerate (3PGA) dehydrogenase. Although NADP had no activity as the electron acceptor, both NADPH and NADH acted as electron donors. Kinetic analyses indicated that the enzyme reaction proceeds via a Theorell-Chance Bi-Bi mechanism. Unlike E. coli PGDH, the S. tokodaii enzyme was not inhibited by l-serine. In addition, both the NAD-dependent 3PGA oxidation and the reverse reaction were enhanced by phosphate and sulfate ions, while NADPH-dependent 3-phosphohydroxypyruvate (PHP) reduction was inhibited. Thus S. tokodaii PGDH appears to be subject to a novel regulatory mechanism not seen elsewhere. A database analysis showed that ST1218 gene forms a cluster with ST1217 gene, and a functional analysis of the ST1217 product expressed in E. coli revealed that it possesses l-glutamate-PHP aminotransferase activity. Taken together, our findings represent the first example of a phosphorylated serine pathway in a hyperthermophilic archaeon.
Shimizu
Yasuhiro
Y
Microbial Genetics Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Sakuraba
Haruhiko
H
Doi
Katsumi
K
Ohshima
Toshihisa
T
D004789:Enzyme Activation; D008970:Molecular Weight; D050543:Phosphoglycerate Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D012694:Serine / Q000096:biosynthesis*; D015398:Signal Transduction / Q000502:physiology; D016944:Sulfolobus / Q000201:enzymology*; D013696:Temperature
D012694:Serine; D050543:Phosphoglycerate Dehydrogenase