All constructs were verified by DNA sequencing. For example, a purified AO from maize (Zea mays) coleoptiles was shown to oxidize indole-3-acetaldehyde and other aliphatic and aromatic aldehydes, including BD (Koshiba et al., 1996). However, at higher temperature and substantial amount of catalyst concentration benzoic acid was obtained along with benzaldehyde. The initial rate was found to increase with increase in concentration of catalyst (18-crown-6) and benzyl alcohol. The pH optimum of AAO4 is 7.0. Analysis of peptide sequences determined in this way identified this protein as encoded by the gene At1g04580. Total RNA (5 μg) was reversed transcribed using oligo(dT) primer and Moloney murine leukemia virus reverse transcriptase (Stratagene). BA-CoA is made from BA, CoA, and ATP in a reaction catalyzed by benzoyl-CoA ligase (BZL) in Arabidopsis (Kliebenstein et al., 2007) as well as in other plants (Beuerle and Pichersky, 2002). Some BA may be derived by degradation of cinnamoyl-CoA through the β-oxidation pathway (Orlova et al., 2006). The reaction usually occurs in two steps where the first step is the oxidation of benzyl alcohol to benzaldehyde and the second step is the formation of benzoic acid from benzaldehyde. Our purification protocol for BD-oxidizing activity identified AAO4 as the protein responsible for most of the activity, consistent with the known high level of expression of its gene in the silique, although we cannot exclude that small amounts of AAO1 (or even AAO2 and AAO3) contribute to this activity. 6). BA was identified by absorption profile and retention time in comparison with an authentic standard. How will you convert the following in not more than two steps : (a) Benzoic acid to Benzaldehyde. 1.5k SHARES. The combined extracts were reduced to dryness under nitrogen. The corresponding cDNA was amplified to yield a 4,014-bp fragment, which was ligated into the vector Topo pEX5-CT/TOPO TA, verified by sequencing, and the recombinant plasmid was transferred into Escherichia coli BL21(DE3)pLysS (Stratagene). How will you bring about the following conversions : Benzoyl chloride to benzaldehyde. Although AAO4 does not possess an NAD-binding site (Schwarz and Mendel, 2006), the addition of NAD+ increased AAO4 activity by about 50%. For determination of kinetic data, we adjusted the total protein quantity to 5 μg per assay. Step 1: Converting benzaldehyde to benzoic acid. The protocol for extraction of BA was modified from Chong et al. How do I convert benzoic acid to benzaldehyde? DEAE Sephadex A25 was swelled in water. How are following conversions carried out ? (iii) 2-bromo-2-methyl butane: Tertiary alkyl halide is most reactive for elimination. Protein extract from Arabidopsis siliques tested for BD-oxidizing activity showed no such activity (data not shown). The seeds of aao4-1 and aao4-2 were found to accumulate just 93% ± 2.0% and 91% ± 2.6% (11.8 ± 1.5 and 11.6 ± 1.52 μmol g−1 seeds) of 3BZO compared with the levels present in wild-type plants (12.7 ± 2.1 μmol g−1 seeds; Fig. The BD-oxidizing activity was enhanced 1.5-fold when 1 mm NAD+ was included in the assay. A semi-empirical model for the reaction was depicted to illustrate the proposed mechanism. 988 Views. Extracts from siliques of aao4-1 and aao4-2 were found to convert BD to BA at a rate of 2.5 ± 0.15 and 2.7 ± 0.15 nkat mg−1 protein, respectively, while the extracts from Arabidopsis wild-type plants did so at a rate that was 1.4-fold higher (3.6 ± 0.15 nkat mg−1 protein; Fig. A mutant that completely lacks benzoylated glucosinolates has recently been found, and the analysis identified the mutated gene as BZL1 (At1g65880; Kliebenstein et al., 2007). Two such glucosinolates, 3-benzoyloxypropylglucosinolate (3BZO) and 4-benzoyloxybutylglucosinolate (4BZO), contain a benzoyl moiety. Benzene was used as the solvent and KMnO4 was taken as the solid reactant. To examine the effect of silencing the AAO4 gene on levels of BA in seeds, we obtained two Arabidopsis ecotype Columbia-0 (Col-0) lines with T-DNA insertions in the AAO4 gene (SALK_047520, designated aao4-1, and SALK_057531, designated aao4-2) and determined that both insertions occur in the second exon of this gene. Write the equations involved in the following reactions :Etard reaction. Question Papers 1851. RT-PCR analysis of mRNA extracted from siliques of wild-type and aao4-1 and aao4-2 homozygous mutant lines. Do the following conversions in not more than two steps :Benzoic acid to benzaldehyde, conversion of the benzoic acid to benzaldehyde. Plants homozygous for a null allele in AAO4 showed a reduction of 30% to 45% in the total levels of BA in seeds as well as 7% to 9% and 32% to 38% decreases in the levels of 3-benzoyloxypropylglucosinolate and 4-benzoyloxybutylglucosinolate, respectively. This protein band, therefore, was excised from the gel and trypsinized, and the resulting peptides were separated by HPLC and analyzed by mass spectrometry. Despite numerous investigations into the synthesis of BA and benzyl alcohol, as well as benzaldehyde (BD), no consensus has yet emerged and no specific enzymes and pathways have been conclusively demonstrated to yield these compounds (for review, see Wildermuth, 2006). The BD-oxidizing activity was extracted from 50 g of Arabidopsis siliques (4–6 weeks old) that were grown under normal conditions, ground in liquid nitrogen with extraction buffer, and stored at −80°C. However, it is clear that the siliques have other enzymes capable of catalyzing this reaction, and it is also equally likely that a substantial amount of BA is derived from a source other than BD. Seo et al. Arabidopsis (Arabidopsis thaliana) siliques synthesize high levels of benzoic acid (BA), which is incorporated into several glucosinolate compounds. After four chromatographic steps (Table I ), the fraction with peak BD oxidase activity exhibited a specific activity of 290 nkat mg−1 protein, with a total increase in specific activity of more than 2,000-fold. Give reason:Ferrimagnetic substances show better magnetism than antiferromagneticsubstances. Since no free BA was detected in the siliques, and the reduction in the level of hydrolyzable BA was similar to the reduction in the levels of 3BZO and 4BZO, it is likely that all of the BA we detected after hydrolysis came from the benzoylated glucosinolates. However with increased catalyst concentration and temperature the formation of benzoic acid along with benzaldehyde has been noted. Identification of the protein responsible for BD-oxidizing activity in Arabidopsis siliques. use DIBAL-H. Or use LiAlH4 to convert acid into benzyl alcohol and then use pcc (pyridinium chloro chromate) or pdc (pyridinium dichromate). ↵1 This work was supported by the National Science Foundation (grant no. All enzyme assays were recorded in triplicate. Protein concentrations were determined by the method of Bradford (1976) using reagents obtained from Bio-Rad. The activation energy and frequency factor of the reaction were found to be 9.149 kJ/mol and 1.85 × 106 respectively. Compound elution was monitored at 200 to 400 nm with a Waters 996 UV/visible photodiode array detector. 988 Views. Benzilic acid was synthesized through a multistep reaction from the starting material of benzaldehyde and through the formations of benzoin and benzil. Protein extracts from Arabidopsis siliques of aao4-1 and aao4-2 showed a decrease BD-oxidizing activity by 30% to 45% as compared with protein extract of wild-type plants (Fig. The enzyme that adds the benzoyl moiety to these glucosinolates in Arabidopsis is most likely an acyltransferase that uses BA-CoA (Graser et al., 2001), although this putative enzyme has not yet been identified. How will you carry out the following conversions? AAO4 transcripts from siliques of plants homozygous for the aao4-1 and aao4-2 alleles could not be detected (Fig. Check you answers with answer keys provided.
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