Detection of Unprecedented CYP74 Enzyme in Mammal: Hydroperoxide Lyase CYP74C44 of the Bat Sturnira hondurensis.
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ABSTRACT:
SUBMITTER: Gorina SS
PROVIDER: S-EPMC9320521 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Hydroperoxide lyases (HPLs) catalyze the splitting of 13S-hydroperoxyoctadecadienoic acid (13S-HPODE) into the green note flavor hexanal and 12-oxo-9(Z)-dodecenoic acid, which is not yet used industrially. Here, HPL from Carica papaya (HPLCP) was cloned and functionally expressed in Escherichia coli to investigate synthesis of 12-oxo-9(Z)-dodecenoic acid in detail. To improve the low catalytic activity of full-length HPLCP, the hydrophobic, non-conserved N-terminal sequence was deleted. This enhanced enzyme activity from initial 10 to 40 U/l. With optimization of solubilization buffer, expression media enzyme activity was increased to 2700 U/l. The tetrameric enzyme was produced in a 1.5 l fermenter and enriched by affinity chromatography. The enzyme preparation possesses a slightly acidic pH optimum and a catalytic efficiency (kcat/KM) of 2.73 × 106 s-1·M-1 towards 13S-HPODE. Interestingly, HPLCP-N could be applied for the synthesis of 12-oxo-9(Z)-dodecenoic acid, and 1 mM of 13S-HPODE was transformed in just 10 s with a yield of 90%. At protein concentrations of 10 mg/ml, the slow formation of the 10(E)-isomer traumatin was observed, pointing to a non-enzymatic isomerization process. Bearing this in mind, a one-pot enzyme cascade starting from safflower oil was developed with consecutive addition of Pseudomonas fluorescens lipase, Glycine max lipoxygenase (LOX-1), and HPLCP-N. A yield of 43% was obtained upon fast extraction of the reaction mixtures after 1 min of HPLCP-N reaction. This work provides first insights into an enzyme cascade synthesis of 12-oxo-9(Z)-dodecenoic acid, which may serve as a bifunctional precursor for bio-based polymer synthesis.
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Project description:A full-length cDNA coding for hydroperoxide lyase (CsHPL) was isolated from cucumber fruits of No. 26 (Southern China type) and No.14-1 (Northern China type), which differed significantly in fruit flavor. The deduced amino acid sequences of CsHPL from both lines show the same and significant similarity to known plant HPLs and contain typical conserved domains of HPLs. The recombinant CsHPL was confirmed to have 9/13-HPL enzymatic activity. Gene expression levels of CsHPL were measured in different organs, especially in fruits of different development stages of both lines. The HPL activities of fruit were identified basing on the catalytic action of crude enzyme extracts incubating with 13-HPOD (13-hydroperoxy-(9Z,12E)-octadecadienoic acid) and 13-HPOD + 9-HPOD (9-hydroperoxy-(10E,12Z)-octadecadienoic acid), and volatile reaction products were analyzed by GC-MS (gas chromatography-mass spectrometry). CsHPL gene expression in No. 26 fruit occurred earlier than that of total HPL enzyme activity and 13-HPL enzyme activity, and that in No. 14-1 fruit was consistent with total HPL enzyme activity and 9-HPL enzyme activity. 13-HPL enzyme activities decreased significantly and the 9-HPL enzyme activities increased significantly with fruit ripening in both lines, which accounted for the higher content of C6 aldehydes at 0-6 day post-anthesis (dpa) and higher content of C9 aldehydes at 9-12 dpa.
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Project description:Bats of the genus Sturnira (Family Phyllostomidae) are characterised by shoulder glands that are more developed in reproductively mature adult males. The glands produce a waxy secretion that accumulates on the fur around the gland, dyeing the fur a dark colour and giving off a pungent odour. These shoulder glands are thought to play a role in their reproductive behaviour. Using gas chromatography-mass spectrometry, we analysed solvent extracts of fur surrounding the shoulder gland in the northern-shouldered bat, Sturnira parvidens to (i) characterise the chemical composition of shoulder gland secretions for the first time, and (ii) look for differences in chemical composition among and between adult males, sub-adult/juvenile males and adult females. Fur solvent extracts were analysed as liquids and also further extracted using headspace solid-phase microextraction to identify volatile components in the odour itself. Odour fingerprint analysis using non-metric multidimensional scaling plots and multivariate analysis revealed clear and significant differences (P < 0.001) between adult males vs both juvenile males and adult females. The chemical components of the shoulder gland secretion included terpenes and phenolics, together with alcohols and esters, most likely derived from the frugivorous diet of the bat. Many of the compounds identified were found exclusively or in elevated quantities among adult (reproductive) males compared with adult females and non-reproductive (juvenile) males. This strongly suggests a specific role in male-female attraction although a function in male-male competition and/or species recognition is also possible.
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Project description:Hydroperoxide lyases (HPLs) catalyze the cleavage of fatty acid hydroperoxides to aldehydes and oxoacids. These volatile aldehydes play a major role in forming the aroma of many plant fruits and flowers. In addition, they have antimicrobial activity in vitro and thus are thought to be involved in the plant defense response against pest and pathogen attack. An HPL activity present in potato leaves has been characterized and shown to cleave specifically 13-hydroperoxides of both linoleic and linolenic acids to yield hexanal and 3-hexenal, respectively, and 12-oxo-dodecenoic acid. A cDNA encoding this HPL has been isolated and used to monitor gene expression in healthy and mechanically damaged potato plants. HPL gene expression is subject to developmental control, being high in young leaves and attenuated in older ones, and it is induced weakly by wounding. HPL enzymatic activity, nevertheless, remains constant in leaves of different ages and also after wounding, suggesting that posttranscriptional mechanisms may regulate its activity levels. Antisense-mediated HPL depletion in transgenic potato plants has identified this enzyme as a major route of 13-fatty acid hydroperoxide degradation in the leaves. Although these transgenic plants have highly reduced levels of both hexanal and 3-hexenal, they show no phenotypic differences compared with wild-type ones, particularly in regard to the expression of wound-induced genes. However, aphids feeding on the HPL-depleted plants display approximately a two-fold increase in fecundity above those feeding on nontransformed plants, consistent with the hypothesis that HPL-derived products have a negative impact on aphid performance. Thus, HPL-catalyzed production of C6 aldehydes may be a key step of a built-in resistance mechanism of plants against some sucking insect pests.
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Project description:Fatty acid hydroperoxides can generate short-chained volatile aldehydes that may participate in plant defence. A grapevine hydroperoxide lyase (VvHPL1) clustering to the CYP74B class was functionally characterized with respect to a role in defence. In grapevine leaves, transcripts of this gene accumulated rapidly to high abundance in response to wounding. Cellular functions of VvHPL1 were investigated upon heterologous expression in tobacco BY-2 cells. A C-terminal green fluorescent protein (GFP) fusion of VvHPL1 was located in plastids. The overexpression lines were found to respond to salinity stress or the bacterial elicitor harpin by increasing cell death. This signal-dependent mortality response was mitigated either by addition of exogenous jasmonic acid or by treatment with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases. By feeding different substrates to recombinantly expressed enzyme, VvHPL1 could also be functionally classified as true 13-HPL. The cognate products generated by this 13-HPL were cis-3-hexenal and trans-2-hexenal. Using a GFP-tagged actin marker line, one of these isomeric products, cis-3-hexenal, was found specifically to elicit a rapid disintegration of actin filaments. This response was not only observed in the heterologous system (tobacco BY-2), but also in a grapevine cell strain expressing this marker, as well as in leaf discs from an actin marker grape used as a homologous system. These results are discussed in the context of a role for VvHPL1 in a lipoxygenase-dependent signalling pathway triggering cell death-related defence that bifurcates from jasmonate-dependent basal immunity.
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Project description:Arabidopsis produces galactolipids containing esters of 12-oxo-phytodienoic acid (OPDA) and dinor-12-oxo-phytodienoic acid (dnOPDA). These lipids are referred to as arabidopsides and accumulate in response to abiotic and biotic stress. We explored the natural genetic variation found in 14 different Arabidopsis accessions to identify genes involved in the formation of arabidopsides. The accession C24 was identified as a poor accumulator of arabidopsides whereas the commonly used accession Col-0 was found to accumulate comparably large amounts of arabidopsides in response to tissue damage. A quantitative trait loci analysis of an F2 population created from a cross between C24 and Col-0 located a region on chromosome four strongly linked to the capacity to form arabidopsides. Expression analysis of HYDROPEROXIDE LYASE 1 (HPL1) showed large differences in transcript abundance between accessions. Transformation of Col-0 plants with the C24 HPL1 allele under transcriptional regulation of the 35S promoter revealed a strong negative correlation between HPL1 expression and arabidopside accumulation after tissue damage, thereby strengthening the view that HPL1 competes with ALLENE OXIDE SYNTHASE (AOS) for lipid-bound hydroperoxide fatty acids. We further show that the last step in the synthesis of galactolipid-bound OPDA and dnOPDA from unstable allene oxides is exclusively enzyme-catalyzed and not the result of spontaneous cyclization. Thus, the results presented here together with previous studies suggest that all steps in arabidopside biosynthesis are enzyme-dependent and apparently all reactions can take place with substrates being esterified to galactolipids.
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