Detection of Unprecedented CYP74 Enzyme in Mammal: Hydroperoxide Lyase CYP74C44 of the Bat Sturnira hondurensis

The genome of the neotropical fruit bat Sturnira hondurensis was recently sequenced, revealing an unexpected gene encoding a plant-like protein, CYP74C44, which shares ca. 90% sequence identity with the putative CYP74C of Populus trichocarpa. The preparation and properties of the recombinant CYP74C44 are described in the present work. The CYP74C44 enzyme was found to be active against the 13- and 9-hydroperoxides of linoleic and α-linolenic acids (13-HPOD, 13-HPOT, 9-HPOD, and 9-HPOT, respectively), as well as the 15-hydroperoxide of eicosapentaenoic acid (15-HPEPE). All substrates studied were specifically transformed into chain cleavage products that are typical for hydroperoxide lyases (HPLs). The HPL chain cleavage reaction was validated by the identification of NaBH4-reduced products (Me/TMS) of 15-HPEPE and 13- and 9-hydroperoxides as (all-Z)-14-hydroxy-5,8,11-tetradecatrienoic, (9Z)-12-hydroxy-9-dodecenoic, and 9-hydroxynonanoic acids (Me/TMS), respectively. Thus, CYP74C44 possessed the HPL activity that is typical for the CYP74C subfamily proteins

Distinct Mechanistic Behaviour of Tomato CYP74C3 and Maize CYP74A19 Allene Oxide Synthases: Insights from Trapping Experiments and Allene Oxide Isolation

The product specificity and mechanistic peculiarities of two allene oxide synthases, tomato LeAOS3 (CYP74C3) and maize ZmAOS (CYP74A19), were studied. Enzymes were vortexed with linoleic acid 9-hydroperoxide in a hexane–water biphasic system (20–60 s, 0 °C). Synthesized allene oxide (9,10-epoxy-10,12-octadecadienoic acid; 9,10-EOD) was trapped with ethanol. Incubations with ZmAOS produced predominantly 9,10-EOD, which was converted into an ethanolysis product, (12Z)-9-ethoxy-10-oxo-12-octadecenoic acid. LeAOS3 produced the same trapping product and 9(R)–α–ketol at nearly equimolar yields. Thus, both α–ketol and 9,10-EOD appeared to be kinetically controlled LeAOS3 products. NMR data for 9,10-EOD (Me) preparations revealed that ZmAOS specifically synthesized 10(E)-9,10-EOD, whereas LeAOS3 produced a roughly 4:1 mixture of 10(E) and 10(Z) isomers. The cyclopentenone cis-10-oxo-11-phytoenoic acid (10-oxo-PEA) and the Favorskii-type product yields were appreciable with LeAOS3, but dramatically lower with ZmAOS. The 9,10-EOD (free acid) kept in hexane transformed into macrolactones but did not cyclize. LeAOS3 catalysis is supposed to produce a higher proportion of oxyallyl diradical (a valence tautomer of allene oxide), which is a direct precursor of both cyclopentenone and cyclopropanone. This may explain the substantial yields of cis-10-oxo-PEA and the Favorskii-type product (via cyclopropanone) with LeAOS3. Furthermore, 10(Z)-9,10-EOD may be produced via the reverse formation of allene oxide from oxyallyl diradical