van Pée Karl-Heinz
Allgemeine Biochemie, TU Dresden, Dresden, Germany.
Methods Enzymol. 2012;516:237-57. doi: 10.1016/B978-0-12-394291-3.00004-6.
Our knowledge about the enzymes catalyzing the incorporation of halide ions during the biosynthesis of halometabolites has increased tremendously during the last 15 years. Between 1960 and 1995, haloperoxidases were the only halogenating enzymes known. However, absolute proof for the connection of haloperoxidases to the biosynthesis of halometabolites is still missing. In 1997, FADH(2)-dependent halogenases were identified as the type of halogenating enzymes responsible for the incorporation of chloride and bromide atoms into aromatic and aliphatic compounds activated for electrophilic attack. FADH(2)-dependent halogenases are two-component systems consisting of a flavin reductase providing the FADH(2) required by the halogenase. Elucidation of the three-dimensional structure of FADH(2)-dependent halogenases led to the understanding of the reaction mechanism, which involves the formation of hypohalous acids. Unactivated carbon atoms were found to be halogenated by nonheme iron, α-ketoglutarate- and O(2)-dependent halogenases. The reaction mechanism of this type of halogenase was shown to involve the formation of a substrate radical. These two types of halogenating enzymes, together with the much less common fluorinases, are the major types of halogenating enzymes. However, the existence of other types of halogenating enzymes, yet not detected, cannot be completely ruled out. Here, we describe the detection, purification, characterization, and reaction mechanisms of flavin-dependent halogenases and of nonheme iron, α-ketoglutarate- and O(2)-dependent halogenases.
在过去15年里，我们对卤代代谢物生物合成过程中催化卤离子掺入的酶的认识有了极大的增长。1960年至1995年间，卤过氧化物酶是唯一已知的卤化酶。然而，卤过氧化物酶与卤代代谢物生物合成之间联系的绝对证据仍然缺失。1997年，依赖FADH(2)的卤化酶被鉴定为负责将氯和溴原子掺入被激活用于亲电攻击的芳香族和脂肪族化合物的卤化酶类型。依赖FADH(2)的卤化酶是双组分系统，由一种黄素还原酶组成，该还原酶提供卤化酶所需的FADH(2)。对依赖FADH(2)的卤化酶三维结构的阐明导致了对反应机制的理解，该机制涉及次卤酸的形成。未活化的碳原子被发现可被非血红素铁、α-酮戊二酸和O(2)依赖的卤化酶卤化。这种类型的卤化酶的反应机制被证明涉及底物自由基的形成。这两种卤化酶类型，连同不太常见的氟化酶，是主要的卤化酶类型。然而，其他尚未被检测到的卤化酶类型的存在也不能完全排除。在这里，我们描述了黄素依赖的卤化酶以及非血红素铁、α-酮戊二酸和O(2)依赖的卤化酶的检测、纯化、表征和反应机制。
