Cloning and expression of pig kidney dopa decarboxylase: comparison of the naturally occurring and recombinant enzymes.
Cloning and expression of pig kidney dopa decarboxylase: comparison of the naturally occurring and recombinant enzymes.
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猪肾多巴脱羧酶的克隆和表达：天然酶和重组酶的比较。
DOI：
10.1042/bj3150249
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
C. Voltattorni
中科院分区：
文献类型：
--
作者：
P. Moore;P. Dominici;C. Voltattorni
L-芳香族氨基酸脱羧酶（多巴脱羧酶; DDC）是一种吡哆醛5 '-磷酸（PLP）依赖性同源二聚体酶，催化L-多巴和其他L-芳香族氨基酸的脱羧。为了推进酶的结构-功能研究，通过将通过文库筛选获得的部分cDNA与通过长寡核苷酸的退火和延伸构建的合成部分连接，克隆了编码来自猪肾的蛋白质的cDNA。然后在大肠杆菌中表达杂合cDNA以产生重组蛋白。在重组酶的表征过程中，意外地观察到它与从猪肾纯化的酶具有某些差异。后者蛋白质每个二聚体结合1个PLP分子，而重组酶被发现每个二聚体结合两个辅酶分子。此外，Vmax是从组织中纯化的蛋白的两倍。在添加底物时，伴随transaldimination的吸光度变化在重组酶中同样大2倍。通过吸光度、CD和荧光光谱法检查各自的脱辅基酶，发现了明显的差异。与猪肾脱辅基酶不同，重组脱辅基蛋白在335 nm处没有显著的吸光度;在后一种情况下，该残留吸光度与正二色性信号相关。当在335 nm处激发时，猪肾脱辅基酶在385 nm处具有明显的发射最大值，与其重组对应物相反，其在约400 nm处显示出弱的宽发射。然而，全酶-脱辅基酶过渡没有显着改变各自的荧光特性的重组或猪肾DDC时，在335 nm处激发。总之，这些发现表明，重组猪肾DDC具有两个活性位点PLP分子，因此显示典型的PLP依赖性同源二聚体酶的结构特征。天然酶含有一个活性位点的PLP分子，而其余的PLP结合位点最有可能被一个非活性的共价结合的辅酶衍生物占据;一些猜测是关于它的起源。重组DDC的辅酶吸收带在335和425 nm处显示出适度的pH依赖性。一个假定的工作模型来解释这种行为。
L-Aromatic amino acid decarboxylase (dopa decarboxylase; DDC) is a pyridoxal 5'-phosphate (PLP)-dependent homodimeric enzyme that catalyses the decarboxylation of L-dopa and other L-aromatic amino acids. To advance structure-function studies with the enzyme, a cDNA that codes for the protein from pig kidney has been cloned by joining a partial cDNA obtained by library screening with a synthetic portion constructed by the annealing and extension of long oligonucleotides. The hybrid cDNA was then expressed in Escherichia coli to produce recombinant protein. During characterization of the recombinant enzyme it was unexpectedly observed that it possesses certain differences from the enzyme purified from pig kidney. Whereas the later protein binds 1 molecule of PLP per dimer, the recombinant enzyme was found to bind two molecules of coenzyme per dimer. Moreover, the Vmax was twice that of the protein purified from tissue. On addition of substrate, the absorbance changes accompanying transaldimination were likewise 2-fold greater in the recombinant enzyme. Examination of the respective apoenzymes by absorbance, CD and fluorescence spectroscopy revealed distinct differences. The recombinant apoprotein has no significant absorbance at 335 nm, unlike the pig kidney apoenzyme; in the latter case this residual absorbance is associated with a positive dichroic signal. When excited at 335 nm the pig kidney apoenzyme has a pronounced emission maximum at 385 nm, in contrast with its recombinant counterpart, which shows a weak broad emission at about 400 nm. However, the holoenzyme-apoenzyme transition did not markedly alter the respective fluorescence properties of either recombinant or pig kidney DDC when excited at 335 nm. Taken together, these findings indicate that recombinant pig kidney DDC has two active-site PLP molecules and therefore displays structural characteristics typical of PLP-dependent homodimeric enzymes. The natural enzyme contains one active-site PLP molecule whereas the remaining PLP binding site is most probably occupied by an inactive covalently bound coenzyme derivative; some speculations are made about its origin. The coenzyme absorbing bands of recombinant DDC show a modest pH dependence at 335 and 425 nm. A putative working model is presented to explain this behaviour.