Tomato CYP74C3 is a Multifunctional Enzyme not only Synthesizing Allene Oxide but also Catalyzing its Hydrolysis and Cyclization

Abstract

The mechanism of the recombinant tomato allene oxide synthase (LeAOS3, CYP74C3) was studied. Incubations of linoleic acid (9S)-hydroperoxide with dilute suspensions of LeAOS3 (10-20 s, 0 degrees C) yield mostly the expected allene oxide (12Z)-9,10-epoxy-10,12-octadecadienoic acid (9,10-EOD), which was detected as its methanol-trapping product. In contrast, the relative yield of 9,10-EOD progressively decreased when the incubations were performed with fourfold, tenfold, or 80-fold larger amounts of LeAOS3, while alpha-ketol and the cyclopentenone rac-cis-10-oxo-11-phytoenoic acid (10-oxo-PEA) became the predominant products. Both the alpha-ketol and 10-oxo-PEA were also produced when LeAOS3 was exposed to preformed 9,10-EOD, which was generated by maize allene oxide synthase (CYP74A). LeAOS3 also converted linoleic acid (13S)-hydroperoxide into the corresponding allene oxide, but with about tenfold lower yield of cyclopentenone. The results indicate that in contrast to the ordinary allene oxide synthases (CYP74A subfamily), LeAOS3 (CYP74C subfamily) is a multifunctional enzyme, catalyzing not only the synthesis, but also the hydrolysis and cyclization of allene oxide.

Determinants governing the CYP74 catalysis: Conversion of allene oxide synthase into hydroperoxide lyase by site‐directed mutagenesis

“…2A). Its identification is substantiated by mass spectral data, coinciding with those described before [15]. This product is formed through the hydrolysis of allene oxide, (12Z)-9,10-epoxy-10,12-octadecadienoic acid [14,15].…”

“…Products (without or with the preliminary NaBH 4 or NaB 2 H 4 reduction) were analyzed as methyl esters/TMS derivatives by GC-MS as described before [15]. Alternatively, products (as methyl esters) were analyzed by the reversed phase radio-HPLC as described before [16].…”

“…The His-tagged recombinant LeAOS3 (WT and mutant forms) were expressed and prepared as described before [14]. His-tagged recombinant proteins were purified by immobilized metal affinity chromatography (IMAC) using the cobalt TALON metal affinity resin (Clontech) as described before [15]. The homogeneity of purified proteins was confirmed by SDS-PAGE.…”

“…The enzyme activity was measured with Varian Cary 50 spectrophotometer by the decrease of 9-HPOD absorbance at 234 nm [15].…”

“…The products were extracted purified with solid phase cartridges, methylated and trimethylsilylated as described before [15], followed by GC-MS analyses. When specified, the products were reduced with NaBH 4 or NaB 2 H 4 after extraction, then methylated and trimethylsilylated.…”