Purification and characterisation of the monodechloroaminopyrrolnitrin 3-halogenase PrnC from pyrrolnitrin biosynthesis

一氯氨基吡咯硝酮3-卤代酶(PRNC)催化吡咯硝酮生物合成的第二步卤化反应。除了色氨酸卤化酶外，直到最近，PRNC一直是已知的唯一一种接受游离底物的黄素依赖的卤化酶。有趣的是，PRNC与色氨酸卤化酶没有显著的氨基酸序列同源性。因此，对依赖黄素的卤代酶进行生化表征，阐明其三维结构，对于理解黄素依赖卤代酶的反应机理和底物专一性具有重要意义。为此目的，将分离PRNC的底物一氯氨基吡咯硝酸甘油酯，并将其用于纯化至均一的PRNC的检测。测定了有机和无机底物的动力学数据(Km、Vmax、kcat)。此外，还将详细分析PRNC的底物专一性和区域选择性。纯化的PrNC将允许阐明三维结构，这反过来又将允许构建突变体来阐明特定氨基酸在PRNC和其他依赖黄素的卤代酶催化的反应中的作用。

Monodechloroaminopyrrolnitrin 3-halogenase (PrnC) catalyses the second halogenation step in pyrrolnitrin biosynthesis. Besides the tryptophan halogenases, PrnC has, until very recently, been the only flavin-dependent halogenase know to accept a free substrate. Interestingly, PrnC has no significant amino acid sequence homology to tryptophan halogenases. Thus, it is of great significance for the understanding of the reaction mechanism and the substrate specificity of flavin-dependent halogenases to characterise PrnC biochemically and to elucidate its 3-dimensional structure. For this purpose, monodechloroaminopyrrolnitrin, the substrate of PrnC, will be isolated and used for the detection of PrnC during purification to homogeneity. The kinetic data (Km, Vmax, kcat) for the organic and inorganic substrates will be determined. In addition, the substrate specificity and the regioselectivity of PrnC will be analysed in detail. Purified PrnC will allow the elucidation of the 3-D structure which in turn will allow the construction of mutants to clarify the role of specific amino acids in the reactions catalysed by PrnC and other flavin-dependent halogenases.