Institute of Biomolecule Reconstruction (iBR), Department of Pharmaceutical Engineering, Sun Moon University, #100 Kalsan-ri, Tangjeong-myeon, Asansi, Chungnam 336-708, Republic of Korea.
Enzyme Microb Technol. 2011 Feb 8;48(2):181-6. doi: 10.1016/j.enzmictec.2010.10.001. Epub 2010 Oct 30.
Cytochrome P450 CYP107AJ1, which was isolated from Streptomyces peucetius and showed high homology with peroxygenases, catalyzed a dealkylation reaction with hydrogen peroxide to provide electrons, protons and oxygen, evading the requirement for a supporting redox protein. Preliminary investigation of its transcriptional level in S. peucetius showed significant expression. Homology modeling and subsequent docking with 7-ethoxycoumarin yielded a reasonable docked structure. cyp107AJ1 cloned into pET28a(+) was expressed in Escherichia coli, and soluble protein was subjected to column-chromatographic purification in order to carry out enzyme assays with 7-ethoxycoumarin. HPLC analysis of the extracted product, corresponding to its LC/MS analysis, showed the dealkylated 7-ethoxycoumarin, which was further established by subsequent GC/MS spectral analysis. We suggest that CYP107AJ1 bypassed the requirement for NAD(P)H and redox partners for generating novel analogues.
细胞色素 P450 CYP107AJ1 是从绛红小单孢菌中分离出来的，与过氧化物酶具有高度同源性，它利用过氧化氢作为电子、质子和氧的供体进行脱烷基反应，从而规避了对辅助氧化还原蛋白的需求。对绛红小单孢菌中其转录水平的初步研究表明其表达水平显著。同源建模和随后与 7-乙氧基香豆素对接得到了一个合理的对接结构。将 cyp107AJ1 克隆到 pET28a(+)中，在大肠杆菌中表达，并通过柱层析纯化可溶蛋白，以进行 7-乙氧基香豆素的酶促反应。提取产物的 HPLC 分析（与其 LC/MS 分析相对应）显示出脱烷基的 7-乙氧基香豆素，随后的 GC/MS 光谱分析进一步证实了这一点。我们认为 CYP107AJ1 绕过了生成新型类似物对 NAD(P)H 和氧化还原伴侣的需求。
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