Seong-Ki Kim

Monocyclic aromatic hydrocarbon degradation by Rhodococcus sp. strain DK17.

ABSTRACT Rhodococcus sp. strain DK17 was isolated from soil and analyzed for the ability to grow on o-xylene as the sole carbon and energy source. Although DK17 cannot grow on m- and p-xylene, it is capable of growth on benzene, phenol, toluene, ethylbenzene, isopropylbenzene, and other alkylbenzene isomers. One UV-generated mutant strain, DK176, simultaneously lost the ability to grow on o-xylene, ethylbenzene, isopropylbenzene, toluene, and benzene, although it could still grow on phenol. The mutant strain was also unable to oxidize indole to indigo following growth in the presence of o-xylene. This observation suggests the loss of an oxygenase that is involved in the initial oxidation of the (alkyl)benzenes tested. Another mutant strain, DK180, isolated for the inability to grow on o-xylene, retained the ability to grow on benzene but was unable to grow on alkylbenzenes due to loss of a meta-cleavage dioxygenase needed for metabolism of methyl-substituted catechols. Further experiments showed that DK180 as well as the wild-type strain DK17 have an ortho-cleavage pathway which is specifically induced by benzene but not by o-xylene. These results indicate that DK17 possesses two different ring-cleavage pathways for the degradation of aromatic compounds, although the initial oxidation reactions may be catalyzed by a common oxygenase. Gas chromatography-mass spectrometry and 300-MHz proton nuclear magnetic resonance spectrometry clearly show that DK180 accumulates 3,4-dimethylcatechol from o-xylene and both 3- and 4-methylcatechol from toluene. This means that there are two initial routes of oxidation of toluene by the strain. Pulsed-field gel electrophoresis analysis demonstrated the presence of two large megaplasmids in the wild-type strain DK17, one of which (pDK2) was lost in the mutant strain DK176. Since several other independently derived mutant strains unable to grow on alkylbenzenes are also missing pDK2, the genes encoding the initial steps in alkylbenzene metabolism (but not phenol metabolism) appear to be present on this approximately 330-kb plasmid.

Identification of a Novel Dioxygenase Involved in Metabolism of o-Xylene, Toluene, and Ethylbenzene by Rhodococcus sp. Strain DK17

ABSTRACT Rhodococcus sp. strain DK17 is able to grow on o-xylene, benzene, toluene, and ethylbenzene. DK17 harbors at least two megaplasmids, and the genes encoding the initial steps in alkylbenzene metabolism are present on the 330-kb pDK2. The genes encoding alkylbenzene degradation were cloned in a cosmid clone and sequenced completely to reveal 35 open reading frames (ORFs). Among the ORFs, we identified two nearly exact copies (one base difference) of genes encoding large and small subunits of an iron sulfur protein terminal oxygenase that are 6 kb apart from each other. Immediately downstream of one copy of the dioxygenase genes (akbA1a and akbA2a) is a gene encoding a dioxygenase ferredoxin component (akbA3), and downstream of the other copy (akbA1b and akbA2b) are genes putatively encoding a meta-cleavage pathway. RT-PCR experiments show that the two copies of the dioxygenase genes are operonic with the downstream putative catabolic genes and that both operons are induced by o-xylene. When expressed in Escherichia coli, AkbA1a-AkbA2a-AkbA3 transformed o-xylene into 2,3- and 3,4-dimethylphenol. These were apparently derived from an unstable o-xylene cis-3,4-dihydrodiol, which readily dehydrates. This indicates a single point of attack of the dioxygenase on the aromatic ring. In contrast, attack of AkbA1a-AkbA2a-AkbA3 on ethylbenzene resulted in the formation of two different cis-dihydrodiols resulting from an oxidation at the 2,3 and the 3,4 positions on the aromatic ring, respectively.

Novel Biosynthetic Pathway of Castasterone from Cholesterol in Tomato

Endogenous brassinosteroids (BRs) in tomato (Lycopersicon esculentum) seedlings are known to be composed of C27- and C28-BRs. The biosynthetic pathways of C27-BRs were examined using a cell-free enzyme solution prepared from tomato seedlings that yielded the biosynthetic sequences cholesterol → cholestanol and 6-deoxo-28-norteasterone ↔ 6-deoxo-28-nor-3-dehydroteasterone ↔ 6-deoxo-28-nortyphasterol → 6-deoxo-28-norcastasterone → 28-norcastasterone (28-norCS). Arabidopsis CYP85A1 that was heterologously expressed in yeast mediated the conversion of 6-deoxo-28-norCS to 28-norCS. The same reaction was catalyzed by an enzyme solution from wild-type tomato but not by an extract derived from a tomato dwarf mutant with a defect in CYP85. Furthermore, exogenously applied 28-norCS restored the abnormal growth of the dwarf mutant. These findings indicate that the C-6 oxidation of 6-deoxo-28-norCS to 28-norCS in tomato seedlings is catalyzed by CYP85, just as in the conversion of 6-deoxoCS to CS. Additionally, the cell-free solution also catalyzed the C-24 methylation of 28-norCS to CS in the presence of NADPH and S-adenosylmethionine (SAM), a reaction that was clearly retarded in the absence of NADPH and SAM. Thus it seems that C27-BRs, in addition to C28-BRs, are important in the production of more active C28-BRs and CS, where a SAM-dependent sterol methyltransferase appears to biosynthetically connect C27-BRs to C28-BRs. Moreover, the tomato cell-free solution converted CS to 26-norCS and [2H6]CS to [2H3]28-norCS, suggesting that C-28 demethylation is an artifact due to an isotope effect. Although previous feeding experiments employing [2H6]CS suggested that 28-norCS was synthesized from CS in certain plant species, this is not supported in planta. Altogether, this study demonstrated for the first time, to our knowledge, that 28-norCS is not synthesized from CS but from cholesterol. In addition, CS and [2H6]CS were not converted into BL and [2H6]BL, respectively, confirming an earlier finding that the active BR in tomato seedlings is not BL but CS. In conclusion, the biosynthesis of 28-norBRs appears to play a physiologically important role in maintaining homeostatic levels of CS in tomato seedlings.

Metabolism of castasterone and brassinolide in mung bean explant

Abstract Different metabolism of brassinolide and its biosynthetic precursor castasterone in explants of mung bean ( Vigna radiata ) seedlings is described. Castasterone was not converted to brassinolide, but to unknown water-soluble metabolites in which major components seemed to be non-glycosidic and minor ones to be glycosidic. It is likely that castasterone is biologically active in its own right without conversion to brassinolide. In contrast, brassinolide was largely converted to 23- O -β- d -glucopyranoside. It seems that 23- O -glycosylation is important in deactivation of brassinosteriods in bean plants.

Identification and Biosynthetic Pathway of Brassinosteroids in Seedling Shoots of Zea mays L.

The potent biosynthetic precursors, 24-methylcholesterol and 24-methylcholestanol, and the endogenous brassinosteroids (BRs), castasterone (CS) and 6-deoxocastasterone (6-deoxoCS), were identified from shoots of maize seedlings. In addition, the presence for activities of several enzymes involved in the late C6-oxida-lion pathway from 24-methylcholestanol to CS was demonstrated in the plants. However, activity for brassinolide (BL) synthase which catalyze the conversion of CS to BL, the last step of the late C6-oxidation pathway, was not detected in the enzyme solution obtained from the maize shoots. Together with the fact that BL was not identified from the maize shoots, these results strongly suggested that BRs in the maize shoots are biosynthesized during seedling growth and the active BR in the shoots is not BL but CS.

The Roles of Phytohormones and AtEXPA3 Gene in Gravitropic Response of Arabidopsis thaliana

We focused on relationship between phytohormones and AtEXPA3 gene in gravitropic response of A. thaliana. RT-PCR analysis shows that AtEXPA3 was highly expressed in actively developing tissues such as leaf, rosette, root and flower tissues. AtEXPA3 gene expression was enhanced by gravistimulation, BR and IAA. Furthermore, decreased gravitropism was observed when treatment of AVG, an ethylene biosynthetic inhibitor, suggesting that ethylene has a gravistimulating effect itself as well as BRs and IAA. Inhibition of gravitropism in AtEXPA3 RNAi mutant suggests that BR, auxin and ethylene are playing roles as regulators of AtEXPA3. In addition, altered gravitropism in BRs signaling mutant (decreased in bri1-301, bak1, and increased BRI-GFP) indicated that BRs signaling mediated the gravitropism. In conclusion, gravitropic responses of Arabidopsis root resulting from root growth were mediated by increased expression of AtEXPA3 gene, which is stimulated by phytohormones.

Identification and Purification of New Brassinosteroid-Conjugates in Arabidopsis thaliana

Metabolism of -castasterone in the presence of -ATP was examined by an enzyme solution prepared from A. thaliana after a reversed phased HPLC, after which a polar metabolite labeled by both and was obtained, suggesting that -CS is phosphorylated by -ATP. To elucidate the structure of the phosphorylated CS, the same enzyme assay was carried out with non-isotopes labeled CS and ATP. In GC-MS analysis the metabolite gave a molecular ion at m/z 664 as a bismethanboronate, suggesting the metabolite is a CS phosphate. Treatment of wheat germ acid phosphatase that hydrolyzed phosphoester bond gave the same mass spectrum and GC retention time in GC-MS analyses, confirming that the metabolite is phosphorylated CS. This is the first example of phosphorylated conjugates of CS in plants.

Importance of C-26 Demethylation for Homeostatic Regulation of Brassinosteroids in Seedling Shoots of Zea mays L

Regulatory mechanism for endogenous levels of castasterone (CS) and its biosynthetic precursors in shoots of maize was investigated by the use of enzyme solution prepared from the plant tissue. When []- and []-CS was used as substrates, []-26-norCS and []-28-norCS were identified as products, indicating that []- and []-CS are differently metabolized into []-26-norCS and []-28-norCS by C-26 and C-28 demethylation, respectively. This suggests that both C-26 and C-28 demethylation can be involved in CS catabolism. In fact that C-28 demethylation only occurred when isotope labeled substrate was used, however, C-26 demethylation is thought be a natural reaction occurred in the maize shoots. When 6-deoxoteasterone (6-deoxoTE) was used, 6-deoxo-26-norTE and 3-dehydro-6-deoxo-26-norTE as well as 6-deoxo-3-dehydroTE and 6-deoxotyphasterol (6-deoxoTY) were identified as enzyme products. When 6-deoxoTY was added, 6-deoxo-26-norTY as well as 6-deoxo-3-dehydroTE and 6-deoxoTE was identified as products. These indicate that C-26 demethylation of 6-deoxoTE, 6-deoxo-3-dehydroTE and 6-deoxoTY as well as a reversible C-3 epimerization from 6-deoxoTE to 6-deoxoTY intermediated by 6-deoxo-3-dehydroTE are operative in the maize shoots, demonstrating that endogenous levels of biosynthetic precursors of CS are also controlled by C-26 demethylation. Therefore, it is thought that C-26 demethylation is an important and a common deactivation process which functions to maintain steady state levels of endogenous brassinosteroids in the maize shoots.