Characterization of the CYP97 and HYD carotene hydroxylase enzymes
Item
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Title
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Characterization of the CYP97 and HYD carotene hydroxylase enzymes
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Identifier
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d_2009_2013:0ecb7c22aabc:11416
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identifier
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11730
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Creator
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Quinlan, Rena Fiona,
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Contributor
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Eleanore T. Wurtzel
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Date
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2012
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Language
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English
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Publisher
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City University of New York.
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Subject
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Molecular biology | Plant sciences
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Abstract
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Escherichia coli functional complementation systems were used to assess rice P450 CYP97A4 (beta-ring hydroxylase) and CYP97C2 (epsilon-ring hydroxylase) as well as maize diiron HYD3 and HYD4 (beta-ring hydroxylases) activities and substrate specificities. Preliminary investigations examining CYP97 enzyme activity only via E. coli complementation showed that the CYP97A4 exhibits major activity toward beta-rings to convert beta-carotene to beta-cryptoxanthin (pathway intermediate) as well as a low amount of zeaxanthin (pathway end-product). In addition, this enzyme exhibited minor activity toward the epsilon-rings of epsilon-epsilon-carotene to convert this substrate to a low amount of lactucaxanthin (pathway end-product). These studies also indicated that the CYP97C2 appeared to be exclusively active toward the epsilon-rings of epsilon-epsilon carotene, converting this substrate to a low amount of lactucaxanthin; no activity toward the beta-rings of beta-carotene was detected for this enzyme.;Subsequent complementation studies tested both individual and combined CYP97/HYD enzyme activities in E. coli accumulating both alpha- and beta-carotene substrates. These studies demonstrated that the CYP97A4 and CYP97C2 enzymes function optimally when expressed together in the conversion of their preferred substrate alpha-carotene to produce lutein. Cells engineered to produce alpha- and beta-carotene and which co-expressed these enzymes generated almost 30% lutein (% total carotenoids); a roughly 10-fold higher amount of lutein relative to zeaxanthin was observed. In contrast, when expressed as individual enzymes the CYP97A4 and CYP97C2 showed suboptimal activity (ie., no lutein produced; only approx. 14%, and 1% of the intermediates zeinoxanthin and alpha-cryptoxanthin generated respectively) regardless of substrate choice. In these cells, the CYP97A4, when expressed alone, preferred the beta-carotene substrate to the alpha-carotene substrate generating a low amount of zeaxanthin (the monohydroxylated intermediate beta-cryptoxanthin accumulated); the CYP97A4 was only moderately active toward the alpha-carotene substrate as only the intermediate zeinoxanthin accumulated (no lutein was produced). When expressed as an individual enzyme, the CYP97C2 was minimally active toward both alpha- and beta-carotene substrates to respectively generate barely detectable amounts of the intermediates alpha-cryptoxanthin and beta-cryptoxanthin.;HYD3 + CYP97C2 and HYD4 + CYP97C2 combinations were also tested using this complementation system. Both enzyme combinations were moderately active toward alpha-carotene, respectively producing low amounts of lutein; although cells co-expressing the HYD3 + CYP97C2 appeared to be somewhat more active toward alpha-carotene than beta-carotene, generating a more than two-fold higher amount of lutein relative to zeaxanthin. When expressed as an individual enzyme, the HYD3 was preferentially active toward beta-carotene to convert this substrate to a low amount of zeaxanthin (end-product); this enzyme was only moderately active toward the alpha-carotene substrate as only the intermediate zeinoxanthin accumulated. The HYD3 exhibited suboptimal activity in our complementation systems whether expressed alone or in combination with the CYP97C2. By contrast, the HYD4 functioned optimally when expressed as an individual enzyme. This enzyme was preferentially active toward beta-carotene and efficiently converted this substrate to zeaxanthin.;This work also examined CYP97/HYD protein localization and protein interaction. In vitro chloroplast import and in vivo GFP fusion assays confirmed that these enzymes are localized to chloroplasts. In addition, import assays were used to determine the suborganellar locations of these enzymes, and in vivo Bimolecular Fluorescence (BiFC) assays were performed to assess protein-protein interaction. Taken together, these studies demonstrated that the CYP97A4 and CYP97C2 enzymes are peripherally-associated to chloroplast membranes where they interact to form a heterodimer complex to function in the efficient conversion of alpha-carotene to lutein. It was expected that these enzymes, which functioned optimally together toward the alpha-carotene substrate in the E. coli complementation system, would interact and localize to the same location in the chloroplast membrane. These data also indicated that the HYD3 and HYD4 enzymes are integrally-bound to the chloroplast membrane where they interact to function in the conversion of beta-carotene to zeaxanthin. (Abstract shortened by UMI.).
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Type
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dissertation
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Source
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2009_2013.csv
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degree
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Ph.D.
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Program
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Biology
