Li Yinxia, Ishida Manabu, Ashida Hiroyuki, Ishikawa Takahiro, Shibata Hitoshi, Sawa Yoshihiro
Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Shimane, Japan.
Biosci Biotechnol Biochem. 2011;75(8):1524-32. doi: 10.1271/bbb.110216. Epub 2011 Aug 7.
We report the molecular characterization and physiological function of a novel L-aspartate dehydrogenase (AspDH). The purified enzyme was a 28-kDa dimeric protein, exhibiting high catalytic activity for L-aspartate (L-Asp) oxidation using NAD and/or NADP as cofactors. Quantitative real-time PCR analysis indicated that the genes involved in the AspDH gene cluster, poly-3-hydroxyalkanoate (PHA) biosynthesis, and the TCA cycle were substantially induced by L-Asp in wild-type cells. In contrast, expression of the aspartase and aspartate aminotransferase genes was substantially induced in the AspDH gene knockout mutant (ΔB3576) but not in the wild type. GC-MS analyses revealed that the wild-type strain synthesized poly-3-hydroxybutyrate from fructose or L-Asp, whereas the ΔB3576 mutant did not synthesize PHA from L-Asp. AspDH gene cluster products might be involved in the biosynthesis of the PHA precursor, revealing that AspDH was a non-NadB type enzyme, and thus entirely different from the previously reported NadB type enzymes working in NAD biosynthesis.
我们报道了一种新型L-天冬氨酸脱氢酶（AspDH）的分子特征和生理功能。纯化后的酶是一种28 kDa的二聚体蛋白，以NAD和/或NADP作为辅因子时，对L-天冬氨酸（L-Asp）氧化表现出高催化活性。定量实时PCR分析表明，野生型细胞中，参与AspDH基因簇、聚3-羟基链烷酸酯（PHA）生物合成以及三羧酸循环（TCA循环）的基因在L-天冬氨酸的作用下被大量诱导。相比之下，天冬氨酸酶和天冬氨酸转氨酶基因的表达在AspDH基因敲除突变体（ΔB3576）中被大量诱导，但在野生型中未被诱导。气相色谱-质谱联用（GC-MS）分析显示，野生型菌株可从果糖或L-天冬氨酸合成聚3-羟基丁酸酯，而ΔB3576突变体不能从L-天冬氨酸合成PHA。AspDH基因簇产物可能参与PHA前体的生物合成，这表明AspDH是一种非NadB型酶，因此与先前报道的参与NAD生物合成的NadB型酶完全不同。
