Analysis of the Cryptophycin P450 Epoxidase Reveals Substrate Tolerance and Cooperativity

Abstract

Cryptophycins are potent anticancer agents isolated from Nostoc sp. ATCC 53789 and Nostoc sp. GSV 224. The most potent natural cryptophycin analogues retain a β-epoxide at the C2′-C3′ position of the molecule. A P450 epoxidase encoded by crpE recently identified from the cryptophycin gene cluster was shown to install this key functional group into cryptophycin-4 (Cr-4) to produce cryptophycin-2 (Cr-2) in a regio-and stereospecific manner. Here we report a detailed characterization of the CrpE epoxidase using an engineered maltose binding protein (MBP)-CrpE fusion. The substrate tolerance of the CrpE polypeptide was investigated with a series of structurally related cryptophycin analogues generated by chemoenzymatic synthesis. The enzyme specifically installed a β-epoxide between C2′ and C3′ of cyclic cryptophycin analogues. The k cat /K m values of the enzyme were determined to provide further insights into the P450 epoxidase catalytic efficiency affected by substrate structural variation. Finally, binding analysis revealed cooperativity of MBP-CrpE toward natural and unnatural desepoxy cryptophycin substrates.

Functional Analysis of an Aspartate‐Based Epoxidation Catalyst with Amide‐to‐Alkene Peptidomimetic Catalyst Analogues

“…[17] Perhaps of great significance, however, is the observation that peptidomimetic catalyst 9 exhibits conformational properties that are surprisingly different from catalyst 5. Whereas catalyst 5 appears as a single conformation in solution by 1 H NMR (400 MHz) (Figure 1 a), consistent with the b-turn observed in the X-ray structure of 8, catalyst 9 appears as a heterogeneous 3.5:1 mixture of two distinct catalyst conformations (Figure 1 b). Notably, the signals coalesce when the NMR sample is heated to 100 8C ([D 6 ]DMSO solvent).…”

“…Whereas the 1 H NMR spectra for catalyst 9 reveal a 3.5:1 conformational mixture (23 8C), fluoroalkene catalyst 11 exhibits an approximately 10:1 mixture of conformations at the same temperature (Figure 1 c). Once again, coalescence of the spectrum is observed when the sample is examined by 1 …”

Chemodiversity in Freshwater and Terrestrial Cyanobacteria – A Source for Drug Discovery

“…The same research group also reported that the thioesterase portion of the crpD synthase could be expressed in a heterologous host, purified, and then reacted in vitro to produce cryptophycin analogs from synthetic, linear precursors [29]. This chemoenzymatic approach was further improved by the identification and purification of the cryptophycin P450 epoxidase, which allowed the chemoenzymatic formation of the benzylic epoxide from styrene-containing cryptophycin precursors [30]. Ultimately, the results from these synthetic and biosynthetic studies could be used to develop a second generation of cryptophycins with improved efficacy with lower toxicity than the first clinical candidate, cryptophycin 52.…”

Cooperative properties of cytochromes P450

“…Cooperativity of cytochromes P450 has been documented in vivo (Tang & Stearns, 2001), in liver microsomes (Oda & Kharasch, 2001; Zhang et al, 2004; Di Marco et al, 2005; Niwa, Murayama, & Yamazaki, 2008a) and in reconstituted systems with purified and isolated individual enzymes, as reviewed in (Guengerich, 1999; Houston & Kenworthy, 2000; Guengerich, 2005; Houston & Galetin, 2005; Atkins, 2006; Tracy, 2006) (Hlavica & Lewis, 2001) (Davydov & Halpert, 2008). In addition to the textbook examples of cooperative cytochromes P450 such as CYP107, CYP3A4, and CYP2C9, cooperativity has also been reported for mammalian xenobiotic metabolizing enzymes CYP1A2 (Sohl et al, 2008), CYP2A6 (Harrelson, Atkins, & Nelson, 2008), CYP2B1 (Scott, He, & Halpert, 2002), CYP2B6, and CYP2E1 (Spatzenegger et al, 2003), for bacterial enzymes CYP102 (Gustafsson et al, 2004; van Vugt-Lussenburg et al, 2006), CYP130 (Ouellet, Podust, & Ortiz de Montellano, 2008), CYP158A2 (Zhao et al, 2005), P450 crpE (Ding et al, 2008), and even for chloroperoxidase (Torres & Aburto, 2005; Aburto, Correa-Basurto, & Torres, 2008). …”