Torres Pazmiño Daniel E, Baas Bert-Jan, Janssen Dick B, Fraaije Marco W
Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Biochemistry. 2008 Apr 1;47(13):4082-93. doi: 10.1021/bi702296k. Epub 2008 Mar 6.
Phenylacetone monooxygenase (PAMO) from Thermobifida fusca is a FAD-containing Baeyer-Villiger monooxygenase (BVMO). To elucidate the mechanism of conversion of phenylacetone by PAMO, we have performed a detailed steady-state and pre-steady-state kinetic analysis. In the catalytic cycle ( k cat = 3.1 s (-1)), rapid binding of NADPH ( K d = 0.7 microM) is followed by a transfer of the 4( R)-hydride from NADPH to the FAD cofactor ( k red = 12 s (-1)). The reduced PAMO is rapidly oxygenated by molecular oxygen ( k ox = 870 mM (-1) s (-1)), yielding a C4a-peroxyflavin. The peroxyflavin enzyme intermediate reacts with phenylacetone to form benzylacetate ( k 1 = 73 s (-1)). This latter kinetic event leads to an enzyme intermediate which we could not unequivocally assign and may represent a Criegee intermediate or a C4a-hydroxyflavin form. The relatively slow decay (4.1 s (-1)) of this intermediate yields fully reoxidized PAMO and limits the turnover rate. NADP (+) release is relatively fast and represents the final step of the catalytic cycle. This study shows that kinetic behavior of PAMO is significantly different when compared with that of sequence-related monooxygenases, e.g., cyclohexanone monooxygenase and liver microsomal flavin-containing monooxygenase. Inspection of the crystal structure of PAMO has revealed that residue R337, which is conserved in other BVMOs, is positioned close to the flavin cofactor. The analyzed R337A and R337K mutant enzymes were still able to form and stabilize the C4a-peroxyflavin intermediate. The mutants were unable to convert either phenylacetone or benzyl methyl sulfide. This demonstrates that R337 is crucially involved in assisting PAMO-mediated Baeyer-Villiger and sulfoxidation reactions.
来自嗜热栖热放线菌的苯丙酮单加氧酶（PAMO）是一种含黄素腺嘌呤二核苷酸（FAD）的拜耳-维利格单加氧酶（BVMO）。为阐明PAMO催化苯丙酮转化的机制，我们进行了详细的稳态和预稳态动力学分析。在催化循环中（kcat = 3.1 s(-1)），烟酰胺腺嘌呤二核苷酸磷酸（NADPH）快速结合（Kd = 0.7 microM），随后4(R)-氢化物从NADPH转移至FAD辅因子（kred = 12 s(-1)）。还原态的PAMO被分子氧快速氧化（kox = 870 mM(-1) s(-1)），生成C4a-过氧黄素。过氧黄素酶中间体与苯丙酮反应形成苄基乙酸酯（k1 = 73 s(-1)）。这一后一动力学事件产生一种酶中间体，我们无法明确确定其归属，可能代表一个Criegee中间体或一种C4a-羟基黄素形式。该中间体相对缓慢的衰变（4.1 s(-1)）产生完全再氧化的PAMO并限制了周转速率。烟酰胺腺嘌呤二核苷酸磷酸（NADP(+)）释放相对较快，代表催化循环的最后一步。本研究表明，与序列相关的单加氧酶，如环己酮单加氧酶和肝微粒体含黄素单加氧酶相比，PAMO的动力学行为有显著差异。对PAMO晶体结构的检查表明，在其他BVMO中保守的残基R337位于靠近黄素辅因子的位置。分析的R337A和R337K突变酶仍能够形成并稳定C4a-过氧黄素中间体。这些突变体无法转化苯丙酮或苄基甲基硫醚。这表明R337在协助PAMO介导的拜耳-维利格反应和硫氧化反应中起关键作用。
