Newton-Vinson P, Hubalek F, Edmondson D E
Departments of Biochemistry and Chemistry, Emory University, Atlanta, Georgia 30322, USA.
Protein Expr Purif. 2000 Nov;20(2):334-45. doi: 10.1006/prep.2000.1309.
The high-level heterologous expression, purification, and characterization of the mitochondrial outer membrane enzyme human liver monoamine oxidase B (MAO B) using the methylotrophic yeast Pichia pastoris expression system are described. A 2-L culture of P. pastoris expresses approximately 1700 U of MAO B activity, with the recombinant enzyme associated tightly with the membrane fraction of the cell lysate. By a modification of the published procedure for purification of bovine liver MAO B [Salach, J. I. (1979) Arch. Biochem. Biophys. 192, 128-137], recombinant human liver MAO B is purified in a 34% yield ( approximately 200 mg from 2 L of cell culture). The isolated enzyme exhibits an M(r) of approximately 60, 000 on SDS-PAGE and 59,474 from electrospray mass spectrometry measurements, which is in good agreement with the mass predicted from the gene sequence and inclusion of the covalent FAD. One mole of covalent FAD per mole of MAO B is present in the purified enzyme and is bound by an 8alpha-S-cysteinyl(397) linkage, as identified by electrospray mass spectrometry of the isolated tryptic/chymotryptic flavin peptide. Recombinant human liver MAO B and bovine liver MAO B are shown to be acetylated at the seryl residues at their respective amino termini. The benzylamine oxidase activity of recombinant MAO B ranges from 3.0 to 3.4 U/mg and steady-state kinetic parameters for this enzyme preparation compare well with those published for the bovine liver enzyme: k(cat) = 600 min(-1), K(m)(benzylamine) = 0.50 mM, and K(m)(O(2)) = 0.33 mM. Kinetic isotope effect parameters using [alpha,alpha-(2)H(2)]benzylamine are also similar to those found for the bovine enzyme. Recombinant MAO B exhibits a (D)k(cat) = 4.7, a (D)[k(cat)/K(m)(benzylamine)] = 4.5, and a (D)[k(cat)/K(m)(O(2))] = 1.0. In contrast to bovine liver MAO B, no evidence was found for the presence of any anionic flavin radical either by UV-vis or by EPR spectroscopy in the resting form of the enzyme. These data demonstrate the successful heterologous expression of a functional, membrane-bound MAO B, which will permit a number of mutagenesis studies as structural and mechanistic probes not previously possible.
本文描述了使用甲基营养酵母毕赤酵母表达系统对线粒体外膜酶人肝脏单胺氧化酶B（MAO B）进行的高水平异源表达、纯化及特性分析。2升毕赤酵母培养物可表达约1700单位的MAO B活性，重组酶紧密结合于细胞裂解物的膜部分。通过改进已发表的牛肝MAO B纯化方法[Salach, J. I. (1979) Arch. Biochem. Biophys. 192, 128 - 137]，重组人肝脏MAO B以34%的产率得到纯化（从2升细胞培养物中约得到200毫克）。分离得到的酶在SDS - PAGE上显示分子量约为60,000，电喷雾质谱测量结果为59,474，这与基因序列预测的质量以及共价结合的FAD相符。纯化后的酶中每摩尔MAO B含有1摩尔共价结合的FAD，通过对分离的胰蛋白酶/胰凝乳蛋白酶黄素肽段的电喷雾质谱鉴定，其通过8α - S - 半胱氨酰（397）键结合。重组人肝脏MAO B和牛肝MAO B在各自氨基末端的丝氨酸残基处均被乙酰化。重组MAO B的苄胺氧化酶活性范围为3.0至3.4单位/毫克，该酶制剂的稳态动力学参数与已发表的牛肝酶参数相当：k(cat) = 600分钟^(-1)，K(m)(苄胺) = 0.50毫摩尔，K(m)(O(2)) = 0.33毫摩尔。使用[α,α - (2)H(2)]苄胺的动力学同位素效应参数也与牛酶的相似。重组MAO B的（D）k(cat) = 4.7，（D）[k(cat)/K(m)(苄胺)] = 4.5，（D）[k(cat)/K(m)(O(2))] = 1.0。与牛肝MAO B不同，无论是通过紫外 - 可见光谱还是电子顺磁共振光谱，均未发现该酶静止形式中存在任何阴离子黄素自由基的证据。这些数据证明了功能性膜结合MAO B的成功异源表达，这将允许进行许多诱变研究，作为以前无法实现的结构和机制探针。
