Copyright © 2020 Elsevier B.V. or its licensors or contributors. The cDNAs and the SALK T-DNA knockout mutant lines of AAO4 (SALK_057531 and SALK_047520) in the Col-0 background were obtained from the Arabidopsis Biological Resource Center. How will you convert the following in not more than two steps : (a) Benzoic acid to Benzaldehyde. The pellet was discarded, the supernatant (300 mg of protein) was passed over DE53 beads (preequilibrated with 50 mm Tris-HCl, pH 7.0) to remove residual impurities, and proteins were eluted with a stepwise KCl gradient (200, 300, and 500 mm KCl in 50 mm Tris-HCl, pH 7.0) with monitoring of enzyme activity. All PCR procedures were performed using KOD DNA polymerase (Novagen) to enhance fidelity. NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. 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). This gene was previously annotated as AAO4 because the protein it encodes shares significant sequence similarity with three aldehyde oxidases encoded by genes AAO1, AAO2, and AAO3 (Koiwai et al., 2000; Mendel and Bittner, 2006). The gene encoding AAO4, At1g04580, is preferentially expressed in developing seeds. Important Solutions 4565. Here, we report that Arabidopsis siliques contain an enzymatic activity capable of converting BD to BA. The divalent cations Mg2+, Fe2+, and Mn2+ as well as EDTA (at 1.0 mm) did not significantly affect enzyme activity, while Cu2+ at this concentration significantly inhibited more than 95% of AAO4 enzyme activity. ABSTRACT. The assays were incubated at 30°C for 30 min, after which the reaction mixture was terminated by the addition of 20 μL of 3.5% (w/v) TCA in 50% (v/v) acetonitrile. Interestingly, BZL1 has a peroxisomal targeting sequence at its C terminus, although the synthesis of benzoylated glucosinolates is believed to be in the cytosol, not the peroxisomes (Chen and Andreasson, 2001). Two such glucosinolates, 3-benzoyloxypropylglucosinolate (3BZO) and 4-benzoyloxybutylglucosinolate (4BZO), contain a benzoyl moiety. Protein concentrations were determined by the method of Bradford (1976) using reagents obtained from Bio-Rad. The BD-oxidizing activity was enhanced 1.5-fold when 1 mm NAD+ was included in the assay. Enzymes that can catalyze the conversion of BD to BA have been reported from bacteria and animals (Yoshida et al., 1998; Hirano et al., 2007). 45. The aromatic metabolites benzoic acid (BA) or benzyl alcohol constitute the backbone of numerous compounds in plants, including taxol and cocaine (Bjorklund and Leete, 1992; Walker and Croteau, 2000), methylbenzoate, and benzylbenzoate (Raguso and Pichersky, 1995; Negre et al., 2003).Despite numerous investigations into the synthesis of BA and benzyl alcohol, as well as benzaldehyde … These findings indicate that in Arabidopsis seeds, oxidation of BD contributes in part to the synthesis of BA. MCB 0331353 to E.P.). Answer. The Km value of AAO4 for BD is comparable to the values reported for Arabidopsis AAO3 and for the maize coleoptile AO (Koshiba et al., 1996; Koiwai et al., 2000). Extracts from 1-, 4-, and 6-week-old siliques converted BD to BA, in the presence of 1 mm NAD+, at a rate of 2.8 to 3.2 nkat mg−1 protein, while similar preparations from mature rosette and cauline leaves did so at rates that were 1.5- to 3-fold lower (Fig. A full-length cDNA of AAO4 was inserted into the bacterial expression vector pEXP5-CT/TOPO, and the recombinant plasmid was transferred to E. coli cells. Total RNA (5 μg) was reverse transcribed using oligo(dT) primer and Moloney murine leukemia virus reverse transcriptase (Stratagene). The oxidation reaction was studied at different degrees of agitation, temperature, catalyst concentration and mole ratio of benzyl alcohol to KMnO4. How would you convert Benzoic acid into Bromobenzene in one step? 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. Do the following conversions in not more than two steps : Benzoic acid to benzaldehyde. Seven unique peptides obtained from this 145-kD protein band (TEIIR, IGVHMEK, GFHPIHK, LPPYNPEK, RINLHTYESLR, QFNVQILNSGHHK, and CDLGFELPVPATMPVVK) matched the protein sequences encoded by AAO4. The 145-kD protein band (Fig. Concept Notes & Videos 736. 2 , lanes 3 and 4) contained several proteins, but the only protein whose concentration (judged by the intensity of staining with Coomassie Brilliant Blue) correlated with BD-oxidizing activity was a 145-kD protein. Practice to excel and get familiar with the paper pattern and the type of questions. 988 Views. ScienceDirect ® is a registered trademark of Elsevier B.V. ScienceDirect ® is a registered trademark of Elsevier B.V. Question Papers 1851. In the presence of magnetic field, the magnetic moments of domains of an antiferromagnetic substances are ordered in such a way half of the magnetic moments are aligned in one direction while the remaining half is in opposite direction.