Kataoka K, Takada H, Tanizawa K, Yoshimura T, Esaki N, Ohshima T, Soda K
Laboratory of Microbial Biochemistry, Kyoto University.
J Biochem. 1994 Oct;116(4):931-6. doi: 10.1093/oxfordjournals.jbchem.a124618.
Phenylalanine dehydrogenase of Thermoactinomyces intermedius acts preferentially on L-phenylalanine and L-tyrosine, whereas leucine dehydrogenase of Bacillus stearothermophilus acts almost exclusively on L-leucine and some other branched-chain L-amino acids. The two enzymes share a sequence similarity (47%). Aiming at elucidation of the mechanism of substrate recognition by the two amino acid dehydrogenases, we have genetically constructed a chimeric enzyme consisting of an N-terminal domain of phenylalanine dehydrogenase containing the substrate-binding region and a C-terminal domain of leucine dehydrogenase containing the NAD(+)-binding region. The chimeric enzyme purified to homogeneity acted on phenylalanine with a specific activity of 6% of that of the parental phenylalanine dehydrogenase and showed a broad substrate specificity in the oxidative deamination, like phenylalanine dehydrogenase. However, it acted much more effectively than phenylalanine dehydrogenase on isoleucine and valine. Its Km values for L-phenylalanine and L-leucine were similar to those of phenylalanine dehydrogenase. The substrate specificity of the chimeric enzyme in the reductive amination was an admixture of those of the two parent enzymes. These results suggest that the two domains of phenylalanine dehydrogenase and leucine dehydrogenase probably can fold independently. Accordingly, their chimera forms a new active enzyme which consists of their N- and C-terminal domains containing the substrate- and coenzyme-binding regions, respectively. However, the two domains of chimeric enzyme interact and communicate with each other to form a new active site and consistently show the new substrate specificity.
中间嗜热放线菌的苯丙氨酸脱氢酶优先作用于L-苯丙氨酸和L-酪氨酸，而嗜热脂肪芽孢杆菌的亮氨酸脱氢酶几乎只作用于L-亮氨酸和其他一些支链L-氨基酸。这两种酶具有47%的序列相似性。为了阐明这两种氨基酸脱氢酶识别底物的机制，我们通过基因工程构建了一种嵌合酶，它由含有底物结合区域的苯丙氨酸脱氢酶N端结构域和含有NAD(+)结合区域的亮氨酸脱氢酶C端结构域组成。纯化至同质的嵌合酶作用于苯丙氨酸，其比活性为亲本苯丙氨酸脱氢酶的6%，并且在氧化脱氨反应中表现出像苯丙氨酸脱氢酶一样的广泛底物特异性。然而，它对异亮氨酸和缬氨酸的作用比苯丙氨酸脱氢酶更有效。其对L-苯丙氨酸和L-亮氨酸的Km值与苯丙氨酸脱氢酶的相似。嵌合酶在还原胺化反应中的底物特异性是两种亲本酶底物特异性的混合。这些结果表明，苯丙氨酸脱氢酶和亮氨酸脱氢酶的两个结构域可能能够独立折叠。因此，它们的嵌合体形成了一种新的活性酶，该酶分别由其含有底物结合区域和辅酶结合区域的N端和C端结构域组成。然而，嵌合酶的两个结构域相互作用并相互通信以形成新的活性位点，并始终表现出新的底物特异性。
