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Purification, molecular cloning, and expression of the gene encoding fatty acid 13‐hydroperoxide lyase from guava fruit ( Psidium guajava )
Author(s) -
Tijet Nathalie,
Wäspi Urs,
Gaskin Duncan J. H.,
Hunziker Peter,
Muller Bernard L.,
Vulfson Evgeny N.,
Slusarenko Alan,
Brash Alan R.,
Whitehead Ian M.
Publication year - 2000
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/s11745-000-0577-z
Subject(s) - complementary dna , biochemistry , enzyme , chemistry , lyase , homotetramer , peptide sequence , linoleic acid , microbiology and biotechnology , fatty acid , biology , gene , protein subunit
Guava fruit was identified as a particularly rich source of 13‐hydroperoxide lyase activity. The enzyme proved stable to chromatographic procedures and was purified to homogeneity. Based on gel filtration and gel electrophoresis, the native enzyme appears to be a homotetramer with subunits of 55 kD. Starting with primers based on the peptide sequence, the enzyme was cloned by polymerase chain reaction with 3′ and 5′ rapid amplification of cDNA ends. The sequence shows approximately 60–70% identity to known 13‐hydroperoxide lyases and is classified in cytochrome P450 74B subfamily as CYP74B5. The cDNA was expressed in Escherichia coli (BL21 cells), with optimal enzyme activity obtained in the absence of isopropyl‐β‐ d ‐thiogalactopyranoside and σ‐aminolevulinic acid. The expressed enzyme metabolized 13( S )‐hydroperoxylinolenic acid over 10‐fold faster than 13( S )‐hydroperoxylinoleic acid and the 9‐hydroperoxides of linoleic and linolenic acids. 13( S )‐Hydroperoxylinolenic acid was converted to 12‐oxododec‐9( Z )‐enoic acid and 3( Z )‐hexenal, as identified by gas chromatography‐mass spectrometry. The turnover number with this substrate, with enzyme concentration estimated from the Soret absorbance, was≈2000/s, comparable to values reported for the related allene oxide synthases. Distinctive features of the guava 13‐hydroperoxide lyase and related cytochrome P450 are discussed.