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  • br Preparation of Recombinant Proteins br Activity Assays


    Preparation of Recombinant Proteins
    Activity Assays The following solvents are used in the procedures described in this section:
    Data Analysis Aryl radical rearrangements typically result in a large number of products being generated by Bmp7. Approaches combining mass spectrometry and NMR spectroscopy for the characterization of polybrominated products generated by Bmp7 have been described previously (Agarwal et al., 2017, Agarwal et al., 2014; Agarwal & Moore, 2014). Oxidative coupling of polyhalogenated phenols and catechols can lead to proton deficient ring systems that are challenging to be unambiguously resolved by NMR experiments (Calcul et al., 2009; Choi, Engene, Smith, Preskitt, & Gerwick, 2010; Liu et al., 2016). In these cases, syntheses of descriptive model compounds and comparison of NMR spectra can be employed for structural assignments (Agarwal et al., 2015). Here, we describe a typical mass spectrometric characterization of products generated by Bmp7 using 2,4-dichlorophenol (13, Fig. 4) as substrate. Mass spectrometry data are generated as described in Section 4.1. Characterization of halogenated products is aided by the natural isotopic distribution of halides. Isotopic distributions of (poly)chlorinated, (poly)brominated, and mixed halogenated molecules have been reviewed previously (Vetter, 2006). Note: The postulated molecular structures shown in Fig. 5, Fig. 6, Fig. 7 are illustrated to demonstrate the use of MS/MS fragmentation in structural assignment of Bmp7 products. Further verification by NMR spectroscopy and other analytical techniques can be guided by the mass spectrometry-based findings.
    Conclusions Protocols described in this chapter utilize P. luteoviolacea 2ta16 derived Bmp9 (ferredoxin) and Bmp10 (ferredoxin-NAD(P)+ oxidoreductase) as the thyrotropin releasing hormone transport partners for the CYP450 enzyme Bmp7 (Table 1, Section 1). Bmp7, as for other CYP450 enzymes, can accept noncognate ferredoxins and ferredoxin-NAD(P)+ oxidoreductases as reaction partners as well. Feeding halophenols to E. coli cultures expressing Bmp7 (but not Bmp9 and Bmp10) leads to the production of coupled products, demonstrating that E. coli ferredoxin(s) and ferredoxin-NAD(P)+ oxidoreductase(s) can substitute Bmp9 and Bmp10 to support the activity of Bmp7 (Agarwal et al., 2014). Furthermore, the Bmp7 active site can accommodate chlorinated, brominated, and iodinated halophenols as substrates. Given the relatively larger steric differences between polybrominated phenols, catechols, and pyrroles, all of which are accepted as substrates by Bmp7, the fact that differentially halogenated phenols such as 6 and 13 are substrates for Bmp7 is perhaps not surprising. A cocrystal structure in complex with polyhalogenated substrate(s) will help describe the molecular bases for the broad substrate tolerance of Bmp7. Crystal structures of CYP450 enzymes that catalyze aryl oxidative radical coupling reactions in complex with their physiological substrates are already available. Two examples include the Streptomyces coelicolor flaviolin dimerizing CYP158A2 and the Mycobacterium tuberculosis CYP121 that installs the intramolecular carbon–carbon bond in cyclodityrosine (Belin et al., 2009; Zhao et al., 2005). Both cocrystal structures demonstrate that only one of the two substrate aryl rings is bound proximal to the iron-porphyrin cofactor while the second aryl ring is bound distal to the cofactor (Fig. 8A and B). Given this steric arrangement, how two aryl radicals are generated within the same CYP450 enzyme active site is presently not clear. Two models have been proposed to address this conundrum. In the first model, radical generated on the aryl substrate bound proximal to the heme cofactor is electronically transferred to the distal aryl ring. A second radical initiation event on the proximal aryl ring then leads to the biradical coupled product (Fig. 8C). In a second proposed model, following radical generation on the proximal aryl ring, the change in the electronic structure leads to a steric switch in which the second aryl ring now moves proximal to the cofactor. Now, the second radical can be generated followed by biradical coupling (Fig. 8D). As both substrate aryl rings for CYP158A2 and CYP121 are identical, distinguishing between the two scenarios is challenging. However, biradical cross coupling of two different aryl substrates 5 and 6 by Bmp7 could perhaps provide an experimentally tractable system to mechanistically distinguish between the two possibilities. In addition to the two biradical mechanisms described earlier, a third possible mechanism could entail radical formation on the heme proximal substrate followed by addition to the distal substrate leading to a radical formation on the distal species which is subsequently quenched by a single-electron transfer event.