Each of the CYP enzymes alluded to above are highly
Each of the CYP450 enzymes alluded to above are highly selective catalysts, in that, the three aryl coupling reactions en route the biosynthesis of 1 are catalyzed by three different CYP450 enzymes in a sequential manner (Fig. 1). In sharp monensin is the recently described marine bacterial CYP450 enzyme Bmp7 that catalyzes the terminal carbon–carbon bond formation en route the biosynthesis of the marine natural product pentabromopseudilin (4, Fig. 2) (Agarwal et al., 2014). The total in vitro reconstitution of the biosynthesis of 4 revealed that the physiological substrates for Bmp7 are 2,3,4-tribromopyrrole (5) and 2,4-dibromophenol (6) (Agarwal et al., 2014; El Gamal, Agarwal, Diethelm, et al., 2016; El Gamal, Agarwal, Rahman, & Moore, 2016). In addition to the communesin core generating CYP450 CnsC, Bmp7 is the only other known CYP450 capable of catalyzing intermolecular crosslinking of two different substrate molecules (Lin et al., 2016). Bmp7 can accept 5 and 6 individually as substrates to catalyze the formation of the marine natural product hexabromo-2,2′-bipyrrole (7), and a suite of dimeric phenolic products coupled via ether and carbon–carbon bonds as illustrated in Fig. 2 (Agarwal et al., 2014). The dimeric phenolic products generated by Bmp7 include 3,3′,5,5′-tetrabromobiphenyl-2,2′-diol (8) and 2-OH-BDE68 (9, BDE68 refers to brominated diphenyl ether congener 68), methoxylated derivatives of which have been shown to accumulate in the blubber of marine mammals (Marsh et al., 2005; Teuten, Xu, & Reddy, 2005). Structures of 4, 7–9 can be rationalized based on the coupling of the rearranged pyrrolic and phenoxy radicals postulated to be generated by Bmp7, followed by rearomatization of the aryl rings (Fig. 2, boxed). In addition to the ortho CO coupled 9, isomeric para CO coupled molecule 10 was observed as a product of the Bmp7 reaction, in addition to CC coupled and para CO coupled products 11 and 12, respectively. Enzymatic synthesis of 11 and 12 involves dehalogenation accompanying oxidative radical coupling, as has been observed previously for halophenols (Dec, Haider, & Bollag, 2003; Osborne, Coggins, Raner, Walla, & Dawson, 2009; Osman, Boeren, Boersma, Veeger, & Rietjens, 1997). Remarkably, the substrate tolerance of Bmp7 extends even further, in that, it can catalyze the formation of dibenzo-p-dioxin products starting from (poly)brominated catechols as substrates (Agarwal & Moore, 2014).
Protocols described in this chapter detail the recombinant expression and purification of the CYP450 Bmp7, and its electron transfer partner proteins Bmp9 (ferredoxin) and Bmp10 (ferredoxin-NAD(P)+ oxidoreductase) from the marine gammaproteobacterium Pseudoalteromonas luteoviolacea 2ta16, together with the NAD(P)H regenerating phosphite dehydrogenase enzyme PtdH (Agarwal et al., 2014; Johannes, Woodyer, & Zhao, 2007). We then describe procedures to assay for the activity of Bmp7 at analytical as well as preparative scales. Owing to the large number of products that Bmp7 generates, which depends on the possible intermediate aryl-radical rearrangements, characterization of its reaction product profile presents an analytical challenge as the chromatographic separation of products and their structure determination using spectroscopic techniques is untenable. To address this challenge, mass spectrometric methods to guide the characterization of Bmp7 products are presented that utilize the unique fragmentation patterns of CC coupled, ortho CO coupled, and para CO coupled halophenols. Using the procedures described here, we demonstrate that Bmp7 can accommodate chlorinated aromatic molecules as substrates, in addition to brominated and iodinated substrates (Agarwal et al., 2014). The broad substrate tolerance of Bmp7 coupled with its ability to generate diverse products makes it an attractive candidate for future engineering efforts to develop a broadly applicable aryl coupling enzyme catalysts.
Equipments and Consumables