Dataset Information

Docking analysis of models for 4-hydroxy-3-methylbut-2-enyl diphosphate reductase and a ferredoxin from Botryococcus braunii, race B.

ABSTRACT: The green microalga Botryococcus braunii Showa, which produces large amounts of triterpene hydrocarbons, exclusively uses the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosyntheses, and the terminal enzyme in this pathway, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR), is regarded as a light-dependent key regulatory enzyme. In order to investigate the possible association of HDR and ferredoxin in this organism, we constructed tertiary structure models of B. braunii HDR (BbHDR) and one of ferredoxin families in the alga, a photosynthetic electron transport F (BbPETF)-like protein, by using counterparts from E. coli and Chlamydomonas reinhardtii as templates, respectively, and performed docking analysis of these two proteins. After docked models are superimposed onto their counterpart proteins in a non-photosynthetic organism, Plasmodium falciparum, the BbPETF-like protein comes in contact with the backside of BbHDR, which was defined in a previous report (Rekittke et al. 2013), and the distance of the two Fe-S centers is 14.7?Å. This distance is in almost the same level as that for P. falicarum, 12.6?Å. To our knowledge, this is the first model suggesting the possible association of HDR with a ferredoxin in O2-evolving photosynthetic organisms.

SUBMITTER: Uchida H

PROVIDER: S-EPMC6879371 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Docking analysis of models for 4-hydroxy-3-methylbut-2-enyl diphosphate reductase and a ferredoxin from Botryococcus braunii, race B.

Uchida Hidenobu H Mizohata Eiichi E Okada Shigeru S

Plant biotechnology (Tokyo, Japan) 20180901 3

The green microalga Botryococcus braunii Showa, which produces large amounts of triterpene hydrocarbons, exclusively uses the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosyntheses, and the terminal enzyme in this pathway, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR), is regarded as a light-dependent key regulatory enzyme. In order to investigate the possible association of HDR and ferredoxin in this organism, we constructed tertiary structure mod ...[more]

PMID: 31819737

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Project description:(E)-4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (IspH or LytB) catalyzes the terminal step of the MEP/DOXP pathway where it converts (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) into the two products, isopentenyl diphosphate and dimethylallyl diphosphate. The reaction involves the reductive elimination of the C4 hydroxyl group, using a total of two electrons. Here we show that the active form of IspH contains a [4Fe-4S] cluster and not the [3Fe-4S] form. Our studies show that the cluster is the direct electron source for the reaction and that a reaction intermediate is bound directly to the cluster. This active form has been trapped in a state, dubbed FeS(A), that was detected by electron paramagnetic resonance (EPR) spectroscopy when one-electron-reduced IspH was incubated with HMBPP. In addition, three mutants of IspH have been prepared and studied, His42, His124, and Glu126 (Aquifex aeolicus numbering), with particular attention paid to the effects on the cluster properties and possible reaction intermediates. None of the mutants significantly affected the properties of the [4Fe-4S](+) cluster, but different effects were observed when one-electron-reduced forms were incubated with HMBPP. Replacing His42 led to an increased K(M) value and a much lower catalytic efficiency, confirming the role of this residue in substrate binding. Replacing the His124 also resulted in a lower catalytic efficiency. In this case, however, the enzyme showed the loss of the [4Fe-4S](+) EPR signal upon addition of HMBPP without the subsequent formation of the FeS(A) signal. Instead, a radical-type signal was observed in some of the samples, indicating that this residue plays a role in the correct positioning of the substrate. The incorrect orientation in the mutant leads to the formation of substrate-based radicals instead of the cluster-bound intermediate complex FeS(A). Replacing the Glu126 also resulted in a lower catalytic efficiency, with yet a third type of EPR signal being detected upon incubation with HMBPP. (31)P and (2)H ENDOR measurements of the FeS(A) species incubated with regular and (2)H-C4-labeled HMBPP reveal that the substrate binds to the enzyme in the proximity of the active-site cluster with C4 adjacent to the site of linkage between the FeS cluster and HMBPP. Comparison of the spectroscopic properties of this intermediate to those of intermediates detected in (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase and ferredoxin:thioredoxin reductase suggests that HMBPP binds to the FeS cluster via its hydroxyl group instead of a side-on binding as previously proposed for the species detected in the inactive Glu126 variant. Consequences for the IspH reaction mechanism are discussed.

Project description:Botryococcus braunii race B is a colony-forming, green algae that accumulates triterpene oils in excess of 30% of its dry weight. The composition of the triterpene oils is dominated by dimethylated to tetramethylated forms of botryococcene and squalene. Although unusual mechanisms for the biosynthesis of botryococcene and squalene were recently described, the enzyme(s) responsible for decorating these triterpene scaffolds with methyl substituents were unknown. A transcriptome of B. braunii was screened computationally assuming that the triterpene methyltransferases (TMTs) might resemble the S-adenosyl methionine-dependent enzymes described for methylating the side chain of sterols. Six sterol methyltransferase-like genes were isolated and functionally characterized. Three of these genes when co-expressed in yeast with complementary squalene synthase or botryococcene synthase expression cassettes resulted in the accumulation of mono- and dimethylated forms of both triterpene scaffolds. Surprisingly, TMT-1 and TMT-2 exhibited preference for squalene as the methyl acceptor substrate, whereas TMT-3 showed a striking preference for botryococcene as its methyl acceptor substrate. These in vivo preferences were confirmed with in vitro assays utilizing microsomal preparations from yeast overexpressing the respective genes, which encode for membrane-associated enzymes. Structural examination of the in vivo yeast generated mono- and dimethylated products by NMR identified terminal carbons, C-3 and C-22/C-20, as the atomic acceptor sites for the methyl additions to squalene and botryococcene, respectively. These sites are identical to those previously reported for the triterpenes extracted from the algae. The availability of closely related triterpene methyltransferases exhibiting distinct substrate selectivity and successive catalytic activities provides important tools for investigating the molecular mechanisms responsible for the specificities exhibited by these unique enzymes.

Dataset Information

Docking analysis of models for 4-hydroxy-3-methylbut-2-enyl diphosphate reductase and a ferredoxin from Botryococcus braunii, race B.

Publications

Docking analysis of models for 4-hydroxy-3-methylbut-2-enyl diphosphate reductase and a ferredoxin from <i>Botryococcus braunii</i>, race B.

Similar Datasets

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