Singer T P, Iankovskaia V L, Bernard S, Cronin C, Sablin S O
Department of Biochemistry and Biophysics, University of California San Francisco 94121, USA.
Vopr Med Khim. 1997 Nov-Dec;43(6):440-56.
An interesting flavoprotein-type monoamine oxidase (MAO) was recently isolated from Aspergillus niger and cloned by Schilling and Lerch (1995a,b) The properties of this MAO, as well as a substantial part of its amino acid sequence resemble those of both MAO A and B from higher animals, raising the possibility that it may be an evolutionary precursor of these mitochondrial enzymes. It differs from MAO A and B in several respect, however, including the fact that it is soluble and of peroxisomal localization and that the FAD is non-covalently attached. We have overexpressed the fungal enzyme (MAO-N) in Escherichia coli, isolated it for the first time in pure form, and, in collaboration with Dr. Elena Sablin, crystallized it. Since several of the observations of previous workers on MAO-N could not be reproduced and seem to be erroneous, we have reexamined its, substrate specificity, interaction with reversible and irreversible inhibitors and other catalytic and molecular properties. MAO-N has a considerably higher turnover number on many aliphatic and aromatic amines than either form of the mammalian enzyme. Some aspects of the substrate specificity resemble those of MAO B, while others are similar to MAO A, including biphasic kinetics in double reciprocal plots. Contrary to the report of Schilling and Lerch (1995a), however, the fungal enzyme does not oxidize serotonin, norepinephrine, dopamine or other biogenic amines. MAO-N is irreversibly inhibited by stoichiometric amounts of both (-)deprenyl and clorgyline in a mechanism-based reaction, forming flavocyanine adducts with N(5) of the FAD, like the mammalian enzymes, but inactivation is much faster with clorgyline than deprenyl, suggesting again a closer resemblance to MAO A than B. The dissociation constants for a large number of reversible competitive inhibitors have been determined for MAO-N and comparison with similar values for MAO A and B again pointed to a much greater similarity to the former than the latter. Experiments designed to change the linkage of the FAD to covalent form by site-directed mutagenesis and to dissociate.
最近，一种有趣的黄素蛋白型单胺氧化酶（MAO）从黑曲霉中分离出来，并由席林和勒奇（1995a，b）克隆。这种MAO的特性以及其大部分氨基酸序列与高等动物的MAO A和B相似，这增加了它可能是这些线粒体酶的进化前体的可能性。然而，它在几个方面与MAO A和B不同，包括它是可溶的且定位于过氧化物酶体，以及FAD是非共价连接的。我们在大肠杆菌中过表达了这种真菌酶（MAO-N），首次以纯形式分离出它，并与埃琳娜·萨布林博士合作使其结晶。由于之前研究人员对MAO-N的一些观察结果无法重现且似乎有误，我们重新研究了它的底物特异性、与可逆和不可逆抑制剂的相互作用以及其他催化和分子特性。MAO-N对许多脂肪族和芳香族胺的周转数比哺乳动物酶的任何一种形式都要高得多。底物特异性的某些方面类似于MAO B，而其他方面则类似于MAO A，包括双倒数图中的双相动力学。然而，与席林和勒奇（1995a）的报告相反，这种真菌酶不氧化血清素、去甲肾上腺素、多巴胺或其他生物胺。MAO-N在基于机制的反应中被化学计量的（-）司来吉兰和氯吉兰不可逆抑制，与哺乳动物酶一样，与FAD的N(5)形成黄素花青素加合物，但氯吉兰的失活速度比司来吉兰快得多，这再次表明它与MAO A比与MAO B更相似。已经确定了MAO-N对大量可逆竞争性抑制剂的解离常数，与MAO A和B的类似值进行比较再次表明，它与前者的相似性远大于与后者的相似性。通过定点诱变改变FAD与共价形式的连接并使其解离的实验。
