Brunelle A, Bi Y A, Lin J, Russell B, Luy L, Berkman C, Cashman J
Seattle Biomedical Research Institute, WA 98109-1651, USA.
Drug Metab Dispos. 1997 Aug;25(8):1001-7.
To examine the possibility for drug metabolism polymorphism, adult human flavin-containing monooxygenases (form 3) (EC 1.14.13.8) that differ at one amino acid were expressed in Escherichia coli as maltose binding protein fusions. The cDNA that was first reported during the cloning of adult human flavin-containing monooxygenase was designated the wild type lys158 enzyme. A second cDNA has been identified as a common polymorphism in some human populations and was designated the glu158 enzyme. The cDNA that encodes both enzymes was subcloned into a high yield protein fusion expression system, expressed, and the protein was partially purified by affinity chromatography and characterized for enzyme activity with selective functional substrate probes. N- and S-oxygenation activity of both enzymes was determined with 10-(N,N-dimethylaminopentyl)-2-(trifluoromethyl)phenothiazine and methyl p-tolyl sulfide, respectively. It was found that expression of both lys158 and glu158 enzymes of the human flavin-containing monooxygenase form 3 as fusions with the maltose binding protein resulted in an enzyme that was soluble and greatly stabilized and had a reduced requirement for detergent during enzyme purification and during the assay for activity. Expression of the fusion proteins has allowed the preparation of stable and highly active enzyme at greater purity than was readily possible in the past. With the exception of the stability and solubility characteristics, the physical and chemical properties of lys158 and glu158 maltose binding fusion proteins of human flavin-containing monooxygenase form 3 variants resembled that of flavin-containing monooxygenase enzyme activity associated with human liver microsomes and enzyme isolated from a previous Escherichia coli expression system that lacked the protein fusion. Comparison of the catalytic activity of the two fusion proteins showed that while both forms were active, there were differences in their substrate specificities. Expression of the adult human flavin-containing monooxygenase form 3 as a maltose binding protein has allowed considerable advances over the previously reported cDNA-expressed enzyme systems and may provide the basis for examining the role of the flavin-containing monooxygenase in human xenobiotic or drug metabolism.
为了研究药物代谢多态性的可能性，在大肠杆菌中表达了一种氨基酸不同的成人含黄素单加氧酶（形式3）（EC 1.14.13.8），作为麦芽糖结合蛋白融合体。在成人含黄素单加氧酶克隆过程中首次报道的cDNA被指定为野生型lys158酶。第二个cDNA已被鉴定为某些人群中的常见多态性，并被指定为glu158酶。编码这两种酶的cDNA被亚克隆到一个高产蛋白融合表达系统中进行表达，然后通过亲和层析对蛋白进行部分纯化，并用选择性功能底物探针表征其酶活性。分别用10-（N，N-二甲基氨基戊基）-2-（三氟甲基）吩噻嗪和对甲苯基甲基硫醚测定两种酶的N-和S-氧化活性。结果发现，将成人含黄素单加氧酶形式3的lys158和glu158酶与麦芽糖结合蛋白融合表达，得到的酶是可溶的，且稳定性大大提高，在酶纯化和活性测定过程中对去污剂的需求降低。融合蛋白的表达使得能够制备出比过去更容易获得的更高纯度的稳定且高活性的酶。除了稳定性和溶解性特征外，成人含黄素单加氧酶形式3变体的lys158和glu158麦芽糖结合融合蛋白的物理和化学性质与人类肝微粒体相关的含黄素单加氧酶活性以及从先前缺乏蛋白融合的大肠杆菌表达系统中分离的酶相似。两种融合蛋白催化活性的比较表明，虽然两种形式都有活性，但它们的底物特异性存在差异。将成人含黄素单加氧酶形式3作为麦芽糖结合蛋白进行表达，相比之前报道的cDNA表达酶系统有了显著进展，可能为研究含黄素单加氧酶在人类异源生物或药物代谢中的作用提供基础。
