RESUMEN
Pyrobaculum islandicum is a hyperthermophilic archaeon that grows optimally at 95-100 °C. In the previous study, we extensively purified a serine racemase from this organism and cloned the gene for overexpression in Escherichia coli (Ohnishi et al. 2008). This enzyme also exhibits highly thermostable L-serine/L-threonine dehydratase activity. In the present study, we aimed to elucidate the molecular mechanisms underlying the high thermostability of this enzyme. A recombinant variant of this enzyme, PiSRvt, constructed by truncating the C-terminal 72 amino acids, was compared with the native enzyme, PiSR. The dehydratase activity of PiSR and PiSRvt was found to owe to a homotrimer and a monomer, respectively, that demonstrated high and moderate thermostability, respectively. These observations reveal that the C-terminal region contributes to monomer trimerization that provides the extreme thermostability.
Asunto(s)
Proteínas Arqueales/química , Racemasas y Epimerasas/química , Termotolerancia , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Estabilidad de Enzimas , Desnaturalización Proteica , Dominios Proteicos , Pyrobaculum/enzimología , Racemasas y Epimerasas/genética , Racemasas y Epimerasas/metabolismoRESUMEN
We have developed a simple, rapid, and inexpensive method of measuring the concentration of intrinsic free D-serine in tissue samples. This method uses chicken D-serine dehydratase in an enzymatic reaction to produce pyruvate, which is detected spectrophotometrically. Pyridoxal 5'-phosphate (PLP), a cofactor of D-serine dehydratase, increased pyruvate formation by 28%. The presence of Zn(2+) or ethylenediaminetetraacetic acid (EDTA) did not have any effect on pyruvate formation under the present assay conditions. In addition, this method was not affected by the presence of a large excess of L-serine, nor by the presence of tissue extracts, and accurately determined concentrations of 2-30 µM (200 pmol-3 nmol) of D-serine. The entire assay requires only 60 min. With this method, we determined the concentration of D-serine in various silkworm tissues. The results were in agreement with high performance liquid chromatography measurements. We found high concentrations of D-serine in silkworm larvae at day 3 of the fifth instar; specifically, 509 nmol g(-1) wet tissue in the midgut, 434 nmol g(-1) in the ovary, and 353 nmol g(-1) in the testis.
