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. 2000 Apr;122(4):1311–1322. doi: 10.1104/pp.122.4.1311

Figure 6.

Figure 6

Reconstitution of recombinant CYP79E1 and CYP79E2 from T. maritima with CYP71E1 from S. bicolor using radiolabeled Tyr as substrate. Spheroblasts were isolated from E. coli cells expressing different constructs of CYP79E1 and CYP79E2, followed by temperature-induced Triton X-114 phase partitioning. Lanes 1 to 6 contain 20 μg protein/reaction mixture. Lane 7 contains 10 μg of protein/reaction mixture. Reconstitution was performed with isolated CYP71E1 and NADPH-Cyt P450 oxidoreductase from S. bicolor. All reconstitutions contain CYP71E1. Lane 1, CYP79E1na, no NADPH; lane 2, CYP79E1na, no NADPH-Cyt P450 oxidoreductase; lane 3, CYP79E1na; lane 4, CYP79E1Δ (1–52)2E1(10aa); lane 5, CYP79E2lacZ(24aa); lane 6, expression vector pSP19g10L; lane 7, S. bicolor CYP79A1Δ (1–33)17α(8aa). Reaction mixtures were analyzed by TLC and the products formed monitored and quantified using a phosphor imager. The position of authentic standards is indicated. Oxime, (E)- and (Z)-p-Hydroxyphenylacetaldoxime; Nitrile, p- hydroxyphenylacetonitrile; Aldehyde, p-hydroxybenzaldehyde.