Metabolism of Hydrocarbon Compounds By Membrane-Bound Metalloenzymes
Date Issued
2014
Date
2014
Author(s)
Chen, Yao-Sheng
Abstract
The membrane metallo-proteins (MMPs) are important catalytic enzyme in organism, particularly the non-heme diiron containing proteins. Membrane-bound fatty acids desaturase superfamily proteins (membrane-FADS superfamily proteins) are classified into type Ⅲ non-heme diiron center proteins that catalyze desaturation and/or monooxygenation in hydrocarbons including aromatics, saturated and unsaturated.
Zebrafish Δ-5/Δ-6 fatty acid desaturase (Z-FADS) catalyzes cascade synthesis of long-chain polyunsaturated fatty acids (LC-PUFAs) and plays pivotal roles in many biological functions. In this study, we deployed the technique of fluorescence resonance energy transfer (FRET) to examine the protein-protein interactions between Z-FADS and cytochrome b5 reductases (CYB5R1-3), elongases 2, 4, 5, 7 (elongation of very long chain fatty acids, ELOVL), respectively and the results indicated that, in endoplasmic reticulum (ER), the Z-FADS can be in close proximity to CYB5R2, 3, and ELOVL protein family, including ELOVL2, 4, 5 and 7, respectively. Furthermore, in the gas chromatography analysis, we proved that the HeLa cells co-transfected with fads, elovl2, 4 and 5 catalyze α-linolenic acid to ω3-series LC-PUFAs, especially the docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Additional data from immuno-fluorescence cytochemistry (IFC) and Western blotting studies demonstrated that Z-FADS also resides in the mitochondria of zebrafish fads transfected HeLa cells. Our results implicated that Z-FADS, the sole fatty acid desaturase ever been identified in zebrafish, can serve as a universal fatty acid desaturase for the whole lipogenesis process.
Xylene monooxygenase (XylM), a non-heme diiron monooxygenase from the prokaryotic system, Pseudomonas putida mt-2, can catalyze toluene to benzyl alcohol and xylene to 4-methylbenzyl alcohol. An E. coli system heterogeneously transformed with part of an TOL operon including the overexpression of XylM and its partner protein of oxidoreductase, XylA, is constructed for its application of whole-cell catalysis. The protein expressions of XylM and XylA in this system are verified by the studies of Western blotting analysis, co-immune-precipitation as well as transmission electron microscopy (TEM) with immune-gold staining experiments. Several substrates including fluorinated butane are designed to study their enzymatic activities. Our results indicated that the aromatic residue(s) within this metalloprotein play important roles for its controlled hydroxylation.
In overall, I exploited both the eukaryotic and prokaryotic membrane-bound non-heme iron proteins including FADS superfamily as my target proteins and pave my way towards understanding the role of Z-FADS on DHA and EPA biosynthesis in Zebrafish as well as showing the promiscuity of hydrophobic pocket in xylene monooxygenase can efficiently achieve selective oxidations in a variety of substrates.
Subjects
金屬酵素
膜結合型脂肪酸去飽和酶超級家族
長鏈多重不飽和脂肪酸
脂肪酸去飽和酶
延長酶
二甲苯單加氧酶
Type
thesis
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