Human biliverdin IXalpha reductase is a zinc-metalloprotein. Characterization of purified and Escherichia coli expressed enzymes.
Human biliverdin IXalpha reductase is a zinc-metalloprotein. Characterization of purified and Escherichia coli expressed enzymes.
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DOI：
10.1111/j.1432-1033.1996.00372.x
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发表时间：
1996
期刊：
影响因子：
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通讯作者：
M. Maines;Bogdan Polevoda;Tian J. Huang;W. McCoubrey
中科院分区：
文献类型：
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作者：
M. Maines;Bogdan Polevoda;Tian J. Huang;W. McCoubrey
胆绿素IX α还原酶（BVR）催化血红素B降解产物胆绿素转化为胆红素。BVR在迄今为止表征的酶中是独特的，因为它具有双重pH/辅因子（NADH、NADPH）特异性。使用通过逆转录和聚合酶链反应从人胎盘RNA产生的探针，从γ文库中分离编码人BVR的cDNA克隆。采用这种方法是因为使用先前分离的大鼠BVR cDNA作为杂交探针的更直接的方法没有成功。对人cDNA进行克隆和测序，结果显示其具有编码296个氨基酸的蛋白质的开放阅读框，其中可以鉴定出先前通过微测序纯化蛋白质鉴定的四种肽。该cDNA与人肾富含poly（A）的RNA中约1.2 kb的单个信息杂交，通过Southern印迹分析，似乎是单拷贝基因的产物。序列分析表明，人类还原酶显示约83%的同一性，在核苷酸和氨基酸水平，与大鼠BVR。在包括羧基末端的一些区域中，蛋白质序列同一性下降至45%。同样值得注意的是，在编码的人还原酶中存在两个额外的半胱氨酸残基（5个，而大鼠为3个）。由其中插入片段与lacZ序列框内克隆的表达质粒产生的蛋白质的特征在于，并表现出双重pH和辅因子依赖性。然而，如动力学分析所示，人酶也可以使用NADH作为辅因子，而大鼠还原酶在生理条件下最有可能仅使用NADPH。蛋白质印迹分析和等电聚焦表明，虽然迁移作为一个单一的带在SDS/PAGE，表达的蛋白质，像从组织中纯化，由几个等电电荷的变体。原子吸收光谱表明，从人肝脏中纯化的蛋白质含有约1：1摩尔比的Zn。通过对纯化的BVR蛋白和在大肠杆菌中表达的融合蛋白进行~（65）Zn交换分析，进一步证实了人BVR是一种Zn金属蛋白。外源性锌也抑制NADPH依赖性，但不是NADH依赖性，活动。因此，NADH和NADPH结合区域的区别在于它们与Zn相互作用的能力;然而，Fe-血卟啉抑制了NADH和NADPH依赖性活性。
Biliverdin IXalpha reductase (BVR) catalyzes the conversion of the heme b degradation product, biliverdin, to bilirubin. BVR is unique among enzymes characterized to date in that it has dual pH/cofactor (NADH, NADPH) specificity. A cDNA clone encoding human BVR was isolated from a gamma library using a probe generated via reverse transcription and the polymerase chain reaction from human placental RNA. This approach was taken because the more direct approach of using the previously isolated rat BVR cDNA as the hybridization probe did not succeed. The human cDNA was cloned and sequenced; it was shown to have an open reading frame encoding a 296-amino-acid protein in which could be identified four peptides previously identified by micro-sequencing purified protein. The cDNA hybridized with a single message of approximately 1.2 kb in human kidney poly(A)-rich RNA, and appeared, by Southern blot analysis, to be the product of a single-copy gene. Sequence analysis indicated that the human reductase shows approximately 83% identity, at both the nucleotide and amino acid levels, with rat BVR. In some regions including the carboxyl terminus, protein sequence identity drops to 45%. Also noteworthy is the presence of two additional cysteine residues in the encoded human reductase (five compared to three for rat). The protein produced by an expression plasmid in which the insert was cloned in frame with lacZ sequences was characterized, and demonstrated dual pH and cofactor dependence. However, as suggested by kinetic analysis, the human enzyme may also use NADH as cofactor, as opposed to the rat reductase, which most likely utilizes only NADPH under physiological conditions. Western blot analysis and isoelectric focusing demonstrate that, although migrating as a single band on SDS/PAGE, the expressed protein, like that purified from tissue, consists of several isoelectric charge variants. Atomic absorption spectroscopy indicates that the protein purified from human liver contains Zn at an approximately 1:1 molar ratio. That human BVR is a Zn metalloprotein was further substantiated by 65Zn exchange analysis of both the purified and the fusion protein expressed in Escherichia coli. Exogenous Zn also inhibits NADPH-dependent, but not NADH-dependent, activity. Hence, the NADH and NADPH binding regions are differentiated by their ability to interact with Zn; Fe-hematoporphyrin, however, inhibited both NADH- and NADPH-dependent activity.