McGuire S M, Silva J C, Casillas E G, Townsend C A
Department of Chemistry, John Hopkins University, Baltimore, Maryland 21218, USA.
Biochemistry. 1996 Sep 3;35(35):11470-86. doi: 10.1021/bi960924s.
The absolute configuration of the dihydrobisfuran ring system characteristic of aflatoxin B1 is essential to the covalent reaction of its metabolically activated form with double-stranded DNA. The biosynthesis of this potent mycotoxin proceeds through three configurationally labile intermediates to racemic versiconal hemiacetal. Subsequent enzymatic cyclization establishes the stereochemistry of this, critical ring fusion in (-)-versicolorin B and is catalyzed by versicolorin B synthase (VBS). The isolation and purification of VBS from Aspergillus parasiticus (SU-1, ATCC 56775) and its kinetic characterization and attempted inactivation are described. Initial purification trials were plagued both by a chromophoric impurity which was difficult to remove and by low recoveries of active protein. The discovery of a remarkably broad pH range of enzyme stability and catalytic activity led to an efficient procedure involving preparative isoelectric focusing and ion exchange FPLC chromatography. The enzyme behaved as a dimer upon gel filtration and migrated with M(r) 78000 Da during denaturing gel electrophoresis. The UV spectrum of pure VBS gave no evidence of a bound chromophore. Detailed kinetic analysis of VBS revealed that this protein selects from two equilibrating enantiomers of versiconal hemiacetal to cyclize the appropriate antipode to optically pure versicolorin B. By varying the amount of enzyme to a fixed concentration of substrate, the rate of enzymic cyclization could be limited by the intrinsic rate of enantiomerization of the substrate under the conditions of reaction. It was possible to quantitate the dynamics of this substrate enantiomerization/cyclization process, to establish the role played by VBS, and to evaluate the significance of each to the overall biosynthesis of aflatoxin. The potential role of an acidic residue of the enzyme in catalysis was supported by analysis of the pH-rate profile of VBS and chemical labeling studies. Successful demonstration of competitive inhibition of VBS by a simple substrate analogue led to the design and synthesis of a potential mechanism-based inactivator of the protein.
黄曲霉毒素B1所特有的二氢双呋喃环系统的绝对构型，对于其代谢活化形式与双链DNA的共价反应至关重要。这种强效霉菌毒素的生物合成通过三种构型不稳定的中间体进行，生成外消旋的Versiconal半缩醛。随后的酶促环化确定了（-）-Versicolorin B中这个关键环融合的立体化学，该过程由Versicolorin B合酶（VBS）催化。本文描述了从寄生曲霉（SU-1，ATCC 56775）中分离和纯化VBS及其动力学表征和失活尝试。最初的纯化试验受到难以去除的发色杂质以及活性蛋白回收率低的困扰。酶稳定性和催化活性的显著宽泛pH范围的发现，导致了一种高效的方法，该方法包括制备性等电聚焦和离子交换FPLC色谱法。该酶在凝胶过滤时表现为二聚体，在变性凝胶电泳中以78000 Da的分子量迁移。纯VBS的紫外光谱没有显示结合发色团的迹象。对VBS的详细动力学分析表明，该蛋白从Versiconal半缩醛的两种平衡对映体中选择，将合适的对映体环化为光学纯的Versicolorin B。通过改变酶的量与固定浓度的底物，在反应条件下，酶促环化速率可能受到底物对映异构化固有速率的限制。可以定量这种底物对映异构化/环化过程的动力学，确定VBS所起的作用，并评估其对黄曲霉毒素整体生物合成的意义。通过对VBS的pH-速率曲线分析和化学标记研究，支持了酶的酸性残基在催化中的潜在作用。成功证明了一种简单底物类似物对VBS的竞争性抑制，从而导致了该蛋白潜在的基于机制的失活剂的设计与合成。
