Humanes L, López-Ruiz A, Merino M T, Roldán J M, Diez J
Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad de Córdoba, Spain.
Appl Environ Microbiol. 1997 Sep;63(9):3385-9. doi: 10.1128/aem.63.9.3385-3389.1997.
Limonoate dehydrogenase from Rhodococcus fascians has been purified to electrophoretic homogeneity by a procedure that consists of ion-exchange, hydrophobic, and affinity chromatography. The native enzyme has a molecular mass of around 128,000 Da and appears to be composed of four similar subunits (30,000 Da each). The isoelectric point is 4.9 as determined by isoelectric focusing. The homogeneous enzyme was used to determine the NH2-terminal amino acid sequence. The enzyme was purified from cells grown in either fructose or limonoate as a carbon source. Limonoate dehydrogenase activity was higher in limonoate-grown cultures. Additionally, the enzyme preparations differed in their affinity for limonoids but not for NAD+. In all cases limonoate dehydrogenase exhibited a higher catalytic rate and stronger affinity for limonoate A-ring lactone than for disodium limonoate, the limonoid traditionally used for in vitro activity assays. Our data confirm previous reports proposing that limonoate A-ring lactone is the physiological substrate for limonoate dehydrogenase. The increase in limonoate dehydrogenase activity observed in limonoate-grown cultures appears to be caused by a rise in protein levels, since chloramphenicol prevented such an effect.
通过离子交换、疏水作用和亲和层析相结合的方法，已将来自叶状红球菌的柠檬苦素脱氢酶纯化至电泳纯。天然酶的分子量约为128,000道尔顿，似乎由四个相似的亚基组成（每个亚基为30,000道尔顿）。通过等电聚焦测定，其等电点为4.9。用纯酶测定了氨基末端氨基酸序列。该酶是从以果糖或柠檬苦素作为碳源生长的细胞中纯化得到的。在以柠檬苦素为碳源生长的培养物中，柠檬苦素脱氢酶的活性更高。此外，酶制剂对柠檬苦素类化合物的亲和力不同，但对NAD⁺的亲和力相同。在所有情况下，柠檬苦素脱氢酶对柠檬苦素A环内酯的催化速率和亲和力均高于传统用于体外活性测定的柠檬苦素二钠盐。我们的数据证实了先前的报道，即柠檬苦素A环内酯是柠檬苦素脱氢酶的生理底物。在以柠檬苦素为碳源生长的培养物中观察到的柠檬苦素脱氢酶活性增加似乎是由蛋白质水平的升高引起的，因为氯霉素可阻止这种效应。
